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Cell Motility and the Cytoskeleton 37:139–148 (1997)
Reactivation of Cell Surface Transport
in Reticulomyxa
Donald D. Orokos,1 Samuel S. Bowser,2,3 and Jeffrey L. Travis1*
1Department
of Biological Sciences, The University at Albany,
State University of New York, Albany
2Department of Biomedical Sciences, The University at Albany,
State University of New York, Albany
3Wadsworth Center, New York State Department of Health, Albany
Granuloreticulosean protists transport particles (e.g., bacteria, algae, and sand
grains) along the outer surfaces of their pseudopodia. This cell surface transport
plays a vital role in feeding, reproduction, shell construction, and locomotion and
can be visualized by the movements of extracellularly adherent polystyrene
microspheres (i.e., latex beads). Our videomicroscopic analyses of transport
associated with the pseudopodia of Reticulomyxa filosa revealed two distinct types
of both intracellular and cell surface transport: (1) saltatory, bidirectional transport
of individual or clustered organelles and/or surface-attached particles, and (2)
continuous, unidirectional bulk or ‘‘resolute’’ motion of aggregated organelles
and/or surface-bound particles. Organelles and surface-attached polystyrene
microspheres remained firmly attached to the microtubule cytoskeletons of
detergent-extracted pseudopodia. Both saltatory and resolute organelle and surface
transport reactivated upon the addition of 0.01–1.0 mM ATP. At 1 mM ATP, the
velocities of reactivated saltatory transport were indistinguishable from those
observed in vivo. The reactivated transport was microtubule-dependent and was
not inhibited by incubation with Ca21-gelsolin under conditions that abolish
rhodamine-phalloidin detection of actin filaments. These findings provide further
support that both intracellular organelle and membrane surface transport are
mediated by a common mechanism, and establish Reticulomyxa as a unique model
system to further study the mechanochemistry of cell surface transport in vitro.
Cell Motil. Cytoskeleton 37:139–148, 1997. r 1997 Wiley-Liss, Inc.
Key words: cell surface transport; Reticulomyxa; organelles
INTRODUCTION
Granuloreticulosean protists, like Reticulomyxa and
the Foraminifera, extend an expansive network of elaborately branched and interconnected pseudopodia called a
reticulopodium (i.e., ‘‘netfoot’’). This distinctive cellular
appendage is built on a scaffold of motile microtubules
that directs its formation and mediates its motility [Travis
et al., 1983]. During these movements, the reticulopodium is rapidly remodeled, resulting in the macro-scale
redistribution of plasma membrane that allows the organism to patrol a relatively large foraging space [reviewed
in Travis and Bowser, 1991]. In addition to these largescale movements, there is considerable evidence for the
r 1997 Wiley-Liss, Inc.
bidirectional transport of plasma membrane domains
along the surface of reticulopodia [Bowser et al., 1984a;
Bowser and Rieder, 1985]. This process, termed cell
surface transport, is involved in many important physiological functions like prey collection and capture [BanContract grant sponsor: The Eppley Foundation for Research. Contract
grant sponsor: National Science Foundation; Contract grant numbers:
OPP92-20146 and MCB 95-05855.
*Correspondence to: Dr. Jeffrey L. Travis, Department of Biological
Sciences, State University of New York at Albany, Albany, NY 12222.
E-mail: jefft@cnsunix.albany.edu
Received 21 November 1996; accepted 7 February 1997
140
Orokos et al.
ner and Culver, 1978], progeny dispersal [Bowser et al.,
1984b], and shell construction [Sandon, 1957; Bowser
and Bernhard, 1993].
Polystyrene microspheres (i.e., latex beads) are
convenient surface markers to study cell surface motility
because they readily attach to and are transported along
the reticulopodial plasma membrane without being internalized [Bowser et al., 1984a]. This approach has yielded
several lines of evidence strongly suggesting that reticulopodial cell surface motility is microtubule-dependent: (1)
particles are transported along the plasma membrane in
close association with the underlying cytoskeletal microtubules [Bowser and Rieder, 1985]; (2) surface-transported microspheres remain associated with the microtubule cytoskeleton even after extraction of the plasma
membrane with non-ionic detergents [Bowser and Rieder,
1985]; (3) distinct structural crossbridges connect the
microtubules to the plasma membrane, and these bridges
are similar to those that link intracellular organelles to the
microtubules [Travis and Bowser, 1988, 1991]. In fact,
the behavior of the cell surface-attached particles so
closely resembles that of intracellularly transported organelles that it has been proposed that a common mechanism
drives both transport processes [Bowser et al., 1984b;
Bowser and Rieder, 1985, 1986; Travis and Bowser,
1991]. In support of this contention, the present paper
demonstrates that cell surface transport can be reactivated
in detergent-lysed motile models of Reticulomyxa under
precisely the same conditions that support the reactivation of microtubule-dependent organelle transport and
microtubule sliding [Koonce and Schliwa, 1986].
MATERIALS AND METHODS
Cell Culture
Reticulomyxa filosa, originally obtained from Dr.
M. Hauser of the University of Bochum, Germany, was
cultured at room temperature in 150 mm diameter Petri
dishes filled with sodium-free Poland Springt water. The
cells were fed with 3–4 grains of plain wheat germ and
subcultured every 4–7 days. On the advice of Prof.
Norbert Hülsmann of the University of Berlin, we now
supplement our cultures with larger algal prey such as
Volvox aureus (obtained from Wards Biological Supply,
Rochester, NY).
Experiments were performed in spring water or
‘‘RM buffer,’’ a solution containing 10 mM Hepes (pH
7.0), 2 mM MgCl2, 0.1 mM MnCl2, and 0.1 mM CaCl2
[Koonce and Schliwa, 1986]. For in vivo observations,
small fragments were cut from cultured organisms and
transferred to Alcian Blue-treated 24 3 60 glass coverslips containing two plastic spacers covered with a fine
bead of vacuum grease. Because Reticulomyxa is multinucleate, these fragments are completely viable and
extend reticulopodia if left undisturbed for a approximately 1 h. After an hour, the fragmented cell body was
surgically removed from the pseudopod to reduce the
amount of cellular debris generated in subsequent treatments. Even without the cell body, the pseudopodia
continue to exhibit rapid intracellular transport and cell
surface motility. A microperfusion chamber was formed
by placing a 22 mm glass coverslip on top of the plastic
spacers.
Cell Surface Labeling
Cell labeling was carried out in microperfusion
chambers under continuous video-enhanced microscopic
observation. Polystyrene microspheres (0.5, 0.8, and 1.07
µm diameter) were obtained from the Sigma Chemical
Co. (St. Louis, MO), resuspended by sonication and
diluted (1:100 or 1:200) into spring water or RM buffer.
Cells were perfused and incubated with this suspension
for 10–20 min.
Reactivation of Cell Surface Transport
Pseudopodia decorated with polystyrene microspheres were lysed for less than 1 min in a solution of 5%
hexylene glycol, 1 mM sodium orthovanadate, and 0.15%
Brij 58 or Triton X-100 in 50% PHEM (30 mM Pipes,
12.5 mM Hepes, 4 mM EGTA, and 1 mM MgCl2 (pH
7.0). The lysed specimens were immediately rinsed in
50% PHEM (pH 7.5) to remove excess vanadate, and
reactivated by perfusion with 1 mM ATP in 50% PHEM
at pH 7.5 [Koonce and Schliwa, 1986; Bowser et al.,
1987]. In some experiments, other nucleotides (ITP, UTP,
CTP, and GTP) at a 1 mM concentration were substituted
for ATP. In other experiments, detergent-extracted specimens were incubated with Ca21, gelsolin, or Ca21-gelsolin
prior to the addition of 1 mM ATP. Actin filament integrity
was assayed by epifluorescence microscopy of lysed, unfixed
specimens stained with tetramethylrhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR).
Video-Enhanced Microscopy and Image
Processing
In all experiments, motility was assayed in only the
peripheral one-third of the pseudopodial network because
this area provided optimal optical conditions. Specimens
were viewed using a Zeiss (Thornwood, NY) IM-35
photomicroscope equipped with differential interference
contrast (DIC) optics. The incident light from a 50 W
Hg-arc burner was passed through serial UV, heat cut, and
narrow band 546 nm interference filters. A Hamamatsu
Photonics (Oak Bridge, IL) C-2400 Newvicon camera
was used for video-DIC microscopy. Digital video processing, including background subtraction, rolling frame
average, and contrast enhancement, was performed with a
Hamamatsu Photonics DVS-3000 image processor. En-
Cell Surface Transport in Reticulomyxa
141
hanced images were recorded on a Panasonic (Matsushita
Electric Industrial Co, Ltd., Osaka, Japan) VHS videocassette recorder and photographed directly from the video
monitor using Kodak (Rochester, NY) T-Max 100 or
T-Max 400 film.
In Vivo and In Vitro Velocity Measurements
Organelle and microsphere velocities were measured from digitally captured frames of the video tape
record. Using a Hamamatsu DVS-3000 Digital Image
Processor (Hamamatsu Photonics), the initial and final
position of individual translocating organelles or microspheres was inscribed with cursors. The distance between
the cursors was then determined using the processor’s
calibrated distance function. A time/date generator
(Thelnors Electronic Lab, Ann Arbor, MI) was used to
measure time in seconds and video fields (1/60 sec).
Organelle and microsphere translocations varied in both
distance and duration. A Student’s t-test for a nondirectional curve was used to determine the statistical significance of velocity differences, whereas a Pearson chisquare test for a two-sided curve was used to determine
the statistical significance of directionality differences.
To examine the relationship between cell surface
translocations of microspheres and intracellularly transported organelles, we examined a total of 166 surface
translocations. These translocations were assigned into
one of three catagories: (1) bidirectional saltations of
individual or small tandem groups of surface particles;
(2) bidirectional saltations of surface-attached particles
moving in tandem with intracellular organelles; or (3)
unidirectional transport of aggregated organelles and
surface-attached microspheres.
Fig. 1. Reticulomyxa transports large algal prey along its reticulopodia.
This low magnification darkfield micrograph shows a portion of a
Reticulomyxa that has captured three Volvox (V) and transported them
along its reticulopodia towards its branched cell body.
TABLE I. Comparison of Organelle and Cell Surface-Attached
Microsphere Transport In Vivoa
Organelle transport
Centripetal
Centrifugal
Surface transport
Centripetal
Centrifugal
Average
rate 6 s.d. (µm/sec)
Number of
observationsb
8.13 6 2.86
7.75 6 2.19
20
20
7.21 6 2.02
6.73 6 1.53
20
21
aCentripetal
transport refers to organelles or microspheres moving
towards the cell body. Centrifugal transport refers to organelles or
microspheres moving away from the cell body.
bTotal number of individual microsphere or organelle translocations
analyzed.
Electron Microscopy
Specimens were fixed at room temperature in 2.0%
glutaraldehyde in 10 mM PIPES, 1 mM EGTA, and 1
mM MgSO4 (pH 7.2) for 1 h. After rinsing, the specimens
were postfixed at 4°C in 0.2% osmium tetroxide and
0.8% potassium ferricyanide in 50 mM Pipes (pH 7.2),
followed by treatment with 0.05% tannic acid [Koonce et
al., 1986]. The cells were then rinsed in distilled water,
dehydrated in ethanol, and prepared for scanning electron
microscopy (SEM). For SEM, fixed cells were dried from
liquid CO2 in a Denton (Cherry Hill, NJ) DV-1 critical
point dryer, sputter coated with 10 nm Au-Pd, and
examined with a Zeiss DSM-940 digital SEM.
RESULTS
Cell Surface Motility In Vivo
Reticulomyxa filosa extended a highly dynamic
pseudopodial network from its multinucleated, ‘‘naked’’
cell body (Fig. 1). When the cells were plated on
cationized glass substrates, pseudopodia became highly
flattened at the most peripheral one-third of the network,
thus making them exceptional specimens for in vivo
observation of microtubule behavior and motility [see
also Chen and Schliwa, 1990]. Bidirectional intracellular
organelle transport occurred throughout the pseudopodia
and these organelles were transported exclusively along
cytoplasmic fibrils (i.e., transport filaments) containing
microtubule bundles [Koonce and Schliwa, 1986; Chen
and Schliwa, 1990]. As seen in Table I, velocities of
centripetal and centrifugal organelle movements did not
differ significantly (P . 0.05).
Like its close relatives the foraminifera, Reticulomyxa transports particles along its pseudopodial surfaces.
Under normal culture conditions, we commonly observed
pseudopodial surface transport of relatively small prey
items such as bacteria or unicellular algae. However, this
protist is also capable of transporting much larger par-
142
Orokos et al.
ticles, like Volvox aureus (approximately 300 µm diameter) seen in Figure 1.
Variously sized (0.5, 0.8, and 1.07 µm) neutral
microspheres, but not negatively charged carboxylated
microspheres, were bound to and transported along
Reticulomyxa pseudopodial surfaces in vivo. We observed two distinct types of pseudopodial surface transport. In the first, individual or small tandem groups of
microspheres displayed bidirectional saltatations. This
transport occurred along even the thinnest pseudopodia,
and microspheres frequently passed close by others that
were moving in the opposite direction (Fig. 2). Motion
analysis revealed that the velocities of centripetal or
centrifugal microsphere saltations (Table I) did not differ
significantly (P . 0.05). Furthermore, surface-attached
microsphere and intracellular organelle saltations displayed a similar average velocity (P . 0.05, Table I) and
directional bias (P . 0.02, Table II). In fact, surfaceattached microspheres and organelles often appeared to
saltate in tandem, as though they were mechanically
linked (Fig. 3). Such linked intracellular and extracellular
motions accounted for 37% of the saltations observed
(Table III).
The second type of cell surface transport involved
the ‘‘resolute’’ (i.e., continuous) movement of irregular
aggregates of surface-attached microspheres and intracellular organelles. These larger aggregates did not move in
a saltatory fashion, but rather moved unidirectionally
(generally towards the cell body) at a constant velocity of
0.5–5.0 µm/sec. Under our typical observation conditions, the pseudopodia displayed predominantly saltatory
surface transport; resolute motion accounted for only 5%
of surface translocations (Table III). Furthermore, the
resolute transport of aggregates was observed typically
only in discrete and localized areas within the network.
However, a withdrawal response that includes resolute
transport of aggregated intracellular organelles and surface-attached microspheres, could be induced throughout
the entire network by exposing Reticulomyxa to noxious
stimuli such as unfiltered high intensity light. When the
stimulus was removed, Reticulomyxa recovered and resumed bidirectional transport and pseudopodial extension.
In favorable preparations we observed that, like
intracellular organelle transport, saltatory and resolute
surface transport occurred along paths defined by cytoplasmic fibrils (data not shown). Microspheres attached to the
pseudopodial surface in regions that were not in contact
with a nearby fibril remained stationary or exhibited
Brownian motion until such a contact was established.
Reactivation of Microsphere Transport
Continuous observation with video-DIC microscopy showed that the vanadate-containing lysis buffer
Fig. 2. Bidirectional surface transport on a single filopodium
A-D: This sequence of four video micrographs shows two microspheres (1 and 2) passing each other in opposite directions while
moving along the pseudopodial surface. The microspheres have
a diameter of 1.07 µm, slightly larger than that of the filopodium.
The three out-of-focus microspheres on the right of the panels
have adsorbed onto the glass substratum; they are used to keep the
panels in register. Panels were photographed from videotape at 1⁄3-sec
intervals.
caused the immediate cessation of all transport, which
was quickly followed by extensive blebbing and sloughing of the plasma membrane and subsequent extraction of
the cytoplasm. However, as noted by previous workers
[Euteneuer et al., 1989], the reticulopodial cytoskeleton
remained intact (Fig. 4). Those microspheres in motion
prior to lysis most frequently remained associated with
the detergent-resistant cytoskeleton; those not in motion
were typically removed with the membrane (Fig. 5).
Furthermore, these motile surface-attached microspheres
remained associated with the cytoskeleton after treatment
with Triton X-100, which completely solubilized membraneous organelles (not illustrated).
Cell Surface Transport in Reticulomyxa
143
TABLE II. Directionality of Organelle and Cell Surface-Attached
Microsphere Transport In Vivoa
Organelle transport
Centripetal
Centrifugal
Surface transport
Centripetal
Centrifugal
Directionality (%)
Number of
observationsb
52
48
560
510
58
42
321
229
aCentripetal
transport refers to organelles or microspheres moving
towards the cell body. Centrifugal transport refers to organelles or
microspheres moving away from the cell body.
bTotal number of individual microsphere or organelle translocations
analyzed.
Saltatory and resolute surface and organelle transport reactivated when ATP was added to lysed networks.
Individual microspheres (or small tandem groups) resumed their bidirectional transport along cytoplasmic
fibrils (Fig. 5C). As was the case in vivo, this surface
particle transport was often accompanied by reactivated
movements of individual organelles. To avoid the possibility that microspheres had adhered non-specifically to
demembranated Reticulomyxa cytoskeletons, we performed all cell lysis and reactivations under continuous
video-DIC observation. Furthermore, we restricted our
motion analysis to those microspheres known to have
been attached to and moving along the cell surface prior
to cell lysis. At 1 mM ATP, the velocities of reactivated
microsphere and organelle saltations were indistinguishable (P . 0.05) (Table IV) from those observed in vivo.
We also noted that the directionality of in vivo particle
movement was conserved in the reactivation system.
Ninety percent (N 5 23) of the reactivated microspheres
resumed moving in the same direction they had been
moving prior to lysis and extraction. Reactivated resolute
transport was characteristically directed towards the cell
body at a constant velocity ranging between 0.5 and
5 µm/sec.
Reactivation of both types of cell surface transport
depended on the addition of ATP. We observed reactivation of surface transport over a range of ATP concentrations from 0.01–1.00 mM, similar to the findings reported
by Schliwa et al. [1991] for intracellular organelle
transport. Other nucleoside triphosphates including GTP,
UTP, CTP, or ITP failed to reactivate surface transport in
the absence of ATP.
Koonce et al. [1986] reported that Reticulomyxa
transport filaments contained bundled microtubules and
actin filaments, but that Ca21-gelsolin had no effect on the
reactivation of organelle transport. We incubated our cell
surface reactivation models with 10 µM Ca21-gelsolin to
disrupt residual actin filaments in the lysed and stabilized
Fig. 3. Surface-attached microspheres can move in tandem with
intracellular organelles. In this video sequence, 0.8-µm-diameter
polystyrene microspheres move along a Reticulomyxa pseudopod in
vivo. A–C: Arrow points to a pair of microspheres moving in tandem
with a pair of organelles (arrowhead ). D: Microspheres and organelles
are no longer associated. Like the transport of organelles, microsphere
transport is saltatory, bidirectional, and follows the same trajectories.
Panels were photographed 1⁄3 sec apart.
cytoskeletal preparations. Despite abolishing F-actin staining with rhodamine-phalloidin, Ca21-gelsolin treatment
had no effect on the reactivation of either intracellular
organelle motility or surface-attached microsphere transport.
144
Orokos et al.
TABLE III. Frequency of Different Modes of Surface Transport
Percent of total
observations (%)
Saltatory transport
Microsphere onlya
Microspheres in tandem with organellesb
Aggregate transportc
58
37
5
aBidirectional
saltations of individual microspheres and organelles
moving independently of each other.
bBidirectional saltations of surface-attached microspheres moving in
tandem with intracellular organelles.
cUnidirectional transport of aggregates of surface-attached microspheres and intracellular organelles.
DISCUSSION
The similarities between organelle and cell surface
transport in Reticulomyxa are striking. Both occur exclusively along intracellular fibrils and both display two
distinct types of transport: saltatory movement of individual particles (Fig. 2) or small tandem groups of
particles (Fig. 3) and resolute transport of aggregated
particles. The velocity profiles of organelle and cell
surface transport saltations did not differ significantly
(Table I). The velocity profiles of resolute organelle and
surface transport were also very similar to each other but
clearly different from those of the saltatory and bidirectional transport (Table II).
The similarities between cell surface and intracellular organelle transport have long been recognized in
foraminifera [Jahn and Rinaldi, 1959; Allen, 1964].
Earlier authors established that organelles and surfaceattached microspheres display the same velocities and
patterns of motility and follow the same transport filaments in pseudopodia [Bowser et al., 1984a; Bowser and
DeLaca, 1985; Bowser and Rieder, 1985; Travis and
Bowser, 1986a]. The quantative analyses of intracellular
and cell surface transport reported here for Reticulomyxa
are in full agreement with this previous work, and are
consistent with the hypothesis that organelle and cell
surface transport share a common mechanism in granuloreticulosean protists [reviewed in Travis and Bowser,
1991].
In Vitro Motility
The cell lysis method employed in the present study
was originally developed by Koonce and Schliwa [1986].
It results in extensive extraction of the pseudopodial
plasma membrane, while leaving the cytoskeleton intact
and fully capable of motility (Fig. 4). Polystyrene microspheres transported on the pseudopodial surface prior to
lysis remain firmly attached to the underlying cytoskeleton even after repeated buffer rinses (Fig. 5B). This
observation is also consistent with past studies with
Allogromia, where cell surface-attached microspheres
and membrane remnants remained bound to the microtubule cytoskeleton by periodic crossbridges after detergent
extraction [Bowser and Rieder, 1985; Travis and Bowser,
1990].
The ATP-dependent reactivation of surface-attached microspheres along the microtubule-based cytoskeleton of lysed Reticulomyxa pseudopodia appears to
be identical to surface motility seen in vivo. First,
individual microspheres resume bidirectional and saltatory transport along the detergent-extracted cytoskeleton
(Fig. 5C). In fact, the great majority of these microspheres resumed moving in the same direction that they
displayed prior to lysis. Resolute surface transport was
also reactivated, confirming observations by Koonce and
Schliwa [1986] of ATP-dependent centripetal motility of
organelle aggregates in vitro.
Mechanism and Regulation of Cell Surface
Transport
The marked similarities between cell surface and
organelle transport in Reticulomyxa strongly suggest that
they share a common mechanism. It would be very
difficult to explain the cell surface movements seen in
Reticulomyxa and its relatives by membrane or lipid flow
[Bretscher, 1976]. This is because the movements are
both saltatory and bidirectional and would require the
shearing of closely apposed and often oppositely directed
phospholipid streams within the membrane. Our detergentdemembranated cytoskeletal preparations clearly lack a
continuous membrane (Fig. 4), conclusively eliminating
lipid or membrane flow, as well as ‘‘surf-riding’’ on
membrane waves [Hewitt, 1979; Ehlers et al., 1996], as
the propulsive force for cell surface transport in Reticulomyxa. Surface-attached microspheres remained associated with the pseudopodial cytoskeleton and resumed
their ATP-dependent movement along organelle-stripped
Triton X-100 cytoskeletons. This observation indicates
that cell surface transport is not propelled by passive
linkage to translocating organelles, and rather suggests
that specific attachments directly link the microspheres to
the cytoskeleton.
Animal cells and amoeboid protists contain a prominent cortical actin-filament cytoskeleton, and myosin
mediated actin-membrane interactions are thought to play
a central role in pseudopodial motility [Luna and Hitt,
1992; Condeelis, 1993; Stossell, 1993; Grebecki, 1994].
However, Reticulomyxa and related protists generally
lack such a conspicuous actin filament cortex, and this
allows their microtubules to closely associate with the
plasma membrane. As such, their surface motility may be
related to that observed in cilia and flagella [Bloodgood,
1988, 1990, 1992; Bloodgood and Salomonsky, 1994],
and not strictly analogous to the directed movement of
glycoproteins and other surface markers on the moving
Cell Surface Transport in Reticulomyxa
145
Fig. 4. Scanning electron micrograph of Brij 58-lysed Reticulomyxa showing surface-attached polystyrene
microspheres (M) and organelles (O) that have remained associated with microtubules following detergent
extraction of pseudopodia. Bar 5 1.0 µm.
lamellipods of vertebrate tissue cells [Sheetz et al., 1989;
Kucick et al., 1989; Hollifield et al., 1990; Heath and
Hollifield, 1991; Forscher et al., 1992].
To date, all indications are that organelle and cell
surface transport in granuloreticuloseans is microtubuledependent [reviewed in Travis and Bowser, 1991]. Koonce
et al. [1986] reported that actin filaments can interdigitate
with microtubules in the transport filaments in Reticulomyxa pseudopodia. However, our observation that cell
surface motility reactivated after incubation with Ca21gelsolin rules out the involvement of actin filaments in
this transport. Schliwa et al. [1991] concluded that a
dynein-like ATPase probably powered both centripetal
and centrifugal organelle transport, although the motor(s)
has not been conclusively identified and characterized at
the molecular level. Recently, we have examined the
mechanochemistry of cell surface transport in lysed and
reactivated Reticulomyxa and found the behavior of the
surface motility ATPase to be virtually identical to that of
the organelle motor [Orokos and Travis, 1995; Orokos et
al., 1996] (manuscript in preparation). Considering the
extensive similarities between cell surface and organelle
transport in this organism, it is likely that they are
powered by the same motor(s) interacting with binding
factors at either the plasma membrane or the organelle
surface. This should not be too surprising because both
types of movement involve the transport of the cytoplasmic side of a membrane interface (either a closed vesicle
or a plasma membrane patch) along the length of a
microtubule. The main difference is that organelles are
transported through a cytoplasmic volume, whereas surface transport is restricted to the two-dimensional plane
of the fluid plasma membrane. It is possible that in
Reticulomyxa, cell surface transport contributes to the
formation and redistribution of new branch pseudopods.
In this regard, the typical branching and anastomoses of
reticulopodia may be likened to the microtubuledependent formation of the branched and anastomosed
intracellular networks of the endoplasmic reticulum
[Dabora and Sheetz, 1988; Dailey and Bridgeman, 1989;
Lee and Chen, 1988; Lee et al., 1989].
One of the best characterized examples of cell
surface motility is that which occurs on ciliary and
flagellar membranes. Bloodgood [1977] showed that the
unicellular alga, Chlamydomonas reinhardtii, transports
146
Orokos et al.
Fig. 5. Microsphere surface transport reactivates in lysed Reticulomyxa. A–C: Sequence of video-enhanced DIC micrographs demonstrating the reactivation of surface-attached microsphere transport. A:
Arrowhead points to a microsphere moving along a pseudopod in vivo.
B: The same region 1 min after lysis in 0.15% Brij-58 (see Materials
and Methods). The arrowhead points to the same microsphere, which
has remained associated with the detergent-resistent cytoskeleton. C:
The same field of view 1 sec after addition of 1.0 mM Mg12-ATP.
Arrowhead points to the microsphere which resumed transport.
TABLE IV. Comparison of Organelle and Cell Surface-Attached
Microsphere Transport In Vitroa
Organelle transport
Centripetal
Centrifugal
Surface transport
Centripetal
Centrifugal
Average
rate 6 s.d. (µm/sec)
Number of
observationsb
7.14 6 2.95
6.30 6 1.98
20
20
7.10 6 3.46
6.02 6 2.68
11
12
aCentripetal
transport refers to organelles or microspheres moving
towards the cell body. Centrifugal transport refers to organelles or
microspheres moving away from the cell body.
bTotal number of microsphere or organelle translocations analyzed.
polystyrene microspheres bidirectionally along its flagellar membranes, and that this flagellar surface motility can
effect a ‘‘gliding’’ mechanism of cell locomotion. Using a
combination of genetic and biochemical approaches, he
and others have identified specific membrane glycoproteins that are also transported along the flagellar membrane [reviewed in Bloodgood, 1990].
The ‘‘9 1 2’’ microtubule axoneme is the major
flagellar cytoskeletal system, and might seem a likely
participant in flagellar surface motility. However, genetic
studies have ruled out the possibility that axonemal
dynein arms and radial spokes are involved [Bloodgood,
1992]. More recently, Kozminski et al. [1995] have
demonstrated that fla10 protein is necessary for the
flagellar surface microsphere transport in Chlamydomonas. Fla10 is a kinesin-like protein, and it has been
localized by electron microscopic immunocytochemistry
to the compartment between the axoneme and the flagellar membrane. Fla 10 is the most attractive candidate for
the Chlamydomonas surface motor, even though its
mechanochemical function has not yet been demonstrated. In addition, these authors showed that mutations
in at least two other loci (FLA 1 and FLA 3) disabled
flagellar surface transport. It is not known how these
genetically implicated proteins interact with each other
and with other transported membrane components such
as the 350 kDa major glycoprotein [Bloodgood, 1990]. It
will also be interesting to determine how widespread
these newly identified flagellar transport proteins are
distributed in other eukaryotes and, in particular, whether
related proteins occur in Reticuloymxa. However, to date
there is little evidence supporting a role for kinesins in
Reticulomyxa transport [Schliwa et al., 1991].
Reticulomyxa and other granuloreticuloseans display a global withdrawal response to noxious stimuli in
which saltatory transport is switched off and centripetal
resolute transport dominates [McGee-Russell and Allen,
1971]. The resolute transport of microspheres and organelles reported here may represent a local activation of this
cytoplasmic withdrawal response. The mechanism of this
switch is not known, but Euteneuer et al. [1989] noted
that addition of cAMP to reactivation buffer caused the
majority of organelles to move in the centripetal direction
in their lysed cell models. They suggested that local
control of directionality may be due to inactivation of
transport in one direction mediated by a protein kinase.
Such a motor regulatory mechanism could explain the
induction of constant velocity unidirectional movements
of individual particles. However, in foraminifera the
withdrawal response also involves complex cytoskeletal
rearrangements, including the transformation of microtubules into aggregates of a helical tubulin polymorph
[Koury et al., 1985; Rupp et al., 1986; Welnhofer and
Travis, 1996]. It remains to be seen whether or not these
helical filaments are associated with the resolute motion
of surface and organelle ‘‘packets’’ in Reticulomyxa.
CONCLUSIONS
ATP-dependent surface transport in Reticulomyxa is
reactivated under the identical conditions that support the
Cell Surface Transport in Reticulomyxa
reactivation of organelle transport. This observation provides compelling support for the contention that organelle
and cell surface transport share a common mechanism.
This should not be too surprising because both involve
the transport of the cytoplasmic side of a membrane
interface (either a closed vesicle or a plasma membrane
patch) along the length of a microtubule.
The present work establishes Reticulomyxa as the
first and, to date, the only system in which cell surface
motility has been reactivated in permeabilized cells. As
such it will be interesting to compare the mechanochemistry of cell surface transport with that of organelles, and
to identify the proteins involved.
ACKNOWLEDGMENTS
This paper is dedicated to Professor Samuel M.
McGee-Russell on the occasion of his retirement from the
Department of Biological Sciences of the University at
Albany. The authors are grateful to Dr. Michael Koonce
for thoughtful advice and instruction during the early
stages of this work. We also thank Drs. Roger Sloboda
and Michael Koonce for critically reading the manuscript. S.S.B. is indebted to Drs. Manfred Schliwa, Ursula
Euteneuer, and Michael Koonce for their hospitality and
for introducing him to Reticulomyxa. This work was
supported by grants from the Eppley Foundation for
Research and the National Science Foundation (OPP
92-20146 awarded to S.S.B. and MCB 95-05855 awarded
to J.L.T.).
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