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: firstname.lastname@example.org 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.  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.  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 . 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  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.  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.  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  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.  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.  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. 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