Cell Motility and the Cytoskeleton 3 5 3 3 1 3 4 4 (1996) F-Actin Network Formation in Tethers and in Pseudopods Stimulated by Chemoattractant Doncho V. Zhelev, Abdullatif M. Alteraifi, and Robert M. Hochmuth Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina Micropipets are used either to deliver a given concentration of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine(fh4LP) to a local region of a human neutrophil or to create a membrane tether. Pseudopods, which have a cylindrical shape and grow at a constant rate, are formed in either case. After reaching a maximum extension, they retract, even in the presence of chemoattractant. As a pseudopod grows, cell granules begin to penetrate the pseudopod region to a “boundary” that defines a distance to the pseudopod’s leading edge that is almost constant. The exclusion of granules from this domain indicates that it is filled with a dense network. The formation of this network involves the plasma membrane because pseudopod growth ceases when a membrane tether is pulled away from the leading edge. The rate of pseudopod growth depends on fMLP concentration just as the number of occupied N-formyl peptide receptors depends on this concentration. The experimental data are explained by assuming that F-actin network is formed next to the plasma membrane. The newly formed network displaces the membrane and the dominant process in the network region then becomes F-actin depolymerization. The rate of pseudopod growth is determined by the rate of the process leading to network formation. This process is apparently an enzymatic type of reaction. It has a positive enthalpy change and, therefore, is endothermic. 0 1996 Wiley-Liss, Inc. Key words: neutrophil, actin, cell activation, motility, MLP, receptor-ligandbinding INTRODUCTION are observed at lower chemoattractant concentrations than the cytotoxic responses [Omann et al., 1987; SnyNeutrophils migrate from the circulation to sites of dennan and Uhing, 19921. In this work we study the inflammation in response to cytokines. First, a circulatdependence of one of the migratory responses, namely ing cell rolls and firmly adheres to the endothelium of the the formation of a pseudopod, on the concentration of the postcapillary venules. Then it transmigrates through the chemoattractant N-formyl-methionyl-leucyl-phenylalagaps in the endothelium to the site of inflammation, nine (fMLP). where it releases enzymes and produces toxic oxygen The migratory responses are mediated by adhesion products. This process is highly regulated and involves receptors [Hendey et al., 1992; Lawson and Maxfield, many specialized molecules, such as adhesion receptors 199.51 and to a great extent by the rearrangement of the [Springer, 19901, cytokine (or chemoattractant) receptors [Snyderman and Uhing, 19921, and cytoskeleton Received May 9, 1996; accepted August 13, 1996. components [Stossel, 19931. The interaction between these specialized molecules is mediated by not yet well- Address reprint requests to Doncho V. Zhelev, Department of Mecharacterized signal transduction pathways [Omann et chanical Engineering and Materials Science, Duke University, al., 1987; Devreotes and Zigmond, 1988; Snyderman Durham, NC 27708-0300. and Uhing, 19921. Regardless of the complexity of this A. M. Alteraifi’s present addresss is Department of Mechanical Enregulation, the neutrophil migratory and cytotoxic re- gineering, United Arab Emirates University, Al-ain, United Arab sponse is dose dependent, where the migratory responses Emirates. 0 1996 Wiley-Liss, Inc. 332 Zhelev et al. cytoskeleton [Stossel, 19931. The cytoskeleton rearrangement includes lamellipodium protrusion and/or pseudopod formation, and cell body contraction [Devreotes and Zigmond, 1988; Evans et al., 19931. Lamellipodium protrusion is of particular interest because it is believed that under certain circumstances (e.g., when cell motility is not altered by the rate of detachment of the adhesion receptors), it determines the rate of crawling [Zigmond, 19931. This protrusion is strongly related to the formation of the F-actin network [Stossel, 19931. The neutrophil’s F-actin has two pools: one is triton soluble and the other is triton insoluble [Watts and Howard, 19931. The triton soluble pool is not stable and depolymerizes in seconds, while the triton insoluble pool is relatively stable and does not depolymerize for tens of minutes. Two pools of F-actin are also found in experiments of actin depolymerization in neutrophil lysates from passive and activated cells [Cano et al., 19911. In this case, one of the pools contains fast depolymerizing F-actin, while the F-actin of the other pool is slow depolymerizing. The fast depolymerizing F-actin is found in activated cells, where it provides almost all F-actin in the lamellipodium region [Cassimeris et al., 19901. The presence of fast depolymerizing F-actin in the lamellipodium region provides conditions for fast F-actin turnover. The turnover is essential for the cytoskeletal rearrangement during cell crawling [Theriot and Mitchison, 19911. However, while the F-actin turnover provides conditions for fast cytoskeleton rearrangement, it is the formation of the network that determines the rate of lamellipodium protrusion and, therefore, the rate of crawling. The formation of F-actin network in human neutrophils stimulated by exposing a local region of the cell body to the chemoattractant fMLP is studied in this work. A new assay is introduced, where fMLP is delivered to a local region of the cell body by a small micropipet. The assay allows a precise measurement of the geometry of the pseudopod region and measurement of the rate of pseudopod extension. These two parameters are used to relate the rate of pseudopod formation to the N L P concentration at different temperatures. The experimental data are explained in terms of the proposed “boundary polymerization model,” where: (1) F-actin network forms at the boundary between the cytoplasm membrane and the leading edge of the advancing network, (2) the newly formed filaments are trapped in the pseudopod region as the boundary of polymerization advances, and (3) the trapped filaments depolymerize providing conditions for F-actin turnover [Theriot and Mitchison, 19911. The chemoattractant stimulated pseudopod protrusion is compared to network formation in tethers [Zhelev and Hochmuth, 19951, which occurs in the absence of chemoattractant. MATERIALS AND METHODS Cell Preparation The preparation of neutrophils has been described elsewhere [Zhelev and Hochmuth, 19951. Briefly, venous blood was drawn from healthy adult donors into vacutainers containing EDTA as an anticoagulant. The blood was centrifuged at 300g for 20 min and the blood plasma collected. The cells from the buffy coat region, which is observed on the top of the packed red cells, were collected. The collected cells were resuspended in the experimental solution. The experimental solution was made of 50% autologous plasma from EDTA vacutainers with 50% Hank’s balanced salt solution (HBSS) without Ca2+ (Sigma Chemical Co., St. Louis, MO). Preparation of Beads and Small Pipets Filled With Chemoattractant Latex beads with immobilized secondary goat antimouse IgG (Sigma) were washed twice in phosphate buffered saline (PBS), resuspended in PBS, vortexed, and sonicated. The beads were incubated at room ternperature for 45 min in PBS containing 1 mg-ml-’ antiLFA- 1p (CD18) monoclonal antibody (mAb) (Becton Dickinson, San Jose, CA). The beads were washed in PBS and resuspended in HBSS. A drop of HBSS containing beads was put in the experimental chamber, where single beads were selected and used for pulling tethers from the neutrophil surface. Small pipets with internal diameters between 1 and 2 pm were filled with a solution made of the solution used in the chamber to which was added the chemoattractant N-formyl-methionyl-leucyl-phenylalanine( N L P , Sigma). The pipets were used to deliver the chemoattractant to a local region of the cell surface and in this way to stimulate the formation of a pseudopod (Fig. 1). Using a pipet allowed a precise control of the amount of chemoattractant and the time of exposure. Micromanipulation The experimental chamber was 2 mm thick and open from both sides to allow micromanipulation. The bottom of the chamber was covered with a coverslip and the top was covered with two parallel glass strips with a 1 mm thick gap. Thermostated water was allowed to flow in the gap for temperature control. The cells were observed with an inverted Leitz (Wetzlar, Germany) microscope with a 100 X oil immersion objective. Micropipets were made from 0.75 mm capillary glass tubing pulled to a fine point with a vertical pipet puller and cut to the desired diameter with a microforge. The pipets were connected to a monometer system, with which the pipet-chamber pressure difference could be controlled between 0.5 and 100 Pa using the micrometer driven F-Actin Network Formation 333 Fig. 1. Formation of a pseudopod stimulated by a local exposure of the cell surface to fh4LP of given concentration. The small pipet is filled with 1 X lo-’ M solution of MLP. A blowing pressure of 20 Pa is applied and the pipet is positioned close the cell surface. Fifteen to 20 s later a pseudopod begins to grow. Both the area of the leading edge and the diameter of the polymerized region remain almost constant as the pseudopod grows. After the pseudopod reaches a maximum extension it starts to retract even though the chemoattractant is still present. The temperature is 22°C. displacement of a water filled reservoir. Suction pressures as high as 40 kPa were obtained with a syringe. The pressures were measured with a differential pressure transducer (Validyne DP15-24). The pressure transducer readings together with a time counter (Vista Electronics Model 401, Vista Electronics, Ramona, CA) were multiplexed on the recorded images. The images were re- corded using a Hamamatsu CCD camera. Distances were measured on the recorded images by using video calipers (Vista Electronics Model 305). Two sets of experiments were performed for measuring the rate of rearrangement of the neutrophil cy- 334 Zhelev et al. toskeleton: experiments where the formation of a pseudopod was stimulated by blowing an FMLP solution from a small pipet (Fig. 1) and experiments where a tether was pulled from the cell surface (Fig. 2). In the first set of experiments a single neutrophil was held in a pipet. Then another pipet filled with a solution of FMLP of a given concentration was positioned 1 to 2 pm from the cell surface. A blowing pressure on the order of 20 Pa was applied and the advancement of the polymerization front for the pseudopod region was measured. The rate of advancement was used to characterize the rate of cytoskeleton rearrangement stimulated by fMLP. During cytoskeleton rearrangement the cortical tension of the cell body changed, while it retained its liquid nature [Evans and Yeung, 19891. This change in the cortical tension was measured by using the holding pipet as described elsewhere [Zhelev and Hochmuth, 19951. Briefly, the cell body was aspirated into the holding pipet until it forms a near hemispherical projection. The projection length of the cell inside the pipet was kept constant by adjusting the pipet suction pressure. The cortical tension T, was calculated by [Evans and Yeung, 19891, T, = Rp - hp where h P was the pipet suction pressure, Rp was the pipet radius, and R,,, was the cell radius outside the pipet. In the presence of a pseudopod the shape of the outside part of the cell body was not quite spherical (Fig. 1). In this case, R,,, represented the outside radius of curvature next to the pipet orifice. This radius of curvature was measured from the recorded images. Usually the pipet radius was significantly smaller than R,,,, so that the imprecise values of R,,, from the recorded images did not affect significantly the calculated value for the cortical tensions. In the second set of experiments (Fig. 2) a single neutrophil was held in a pipet. A bead with a monoclonal antibody (mAb) for the CD18 adhesion receptor was brought into contact with the cell surface for 3 to 5 s and then withdrawn from the cell surface to a distance of 10 to 15 pm. During contact one or several CD18 receptors bound to their antibodies on the bead surface. When the bead is withdrawn, the bound CD18 receptors pulled the membrane from the cell and one or several tethers were formed. A polymer formed in the tether, beginning at the cell body and spreading up the tether until a pseudopodlike structure was formed [Zhelev and Hochmuth, 19951. The rate of advancement of the polymerization front along the tether was used as a measure of the rate of cytoskeleton rearrangement in the absence of chemoattractant. Only cells with single tethers were used in the experiments. RESULTS FMLP Stimulated Pseudopod Formation In the experiment illustrated in Figure 1, the exposure of a local region of the cell surface to a fMLP gradient induces the formation of a single pseudopod. During this process the cell cytoplasm undergoes a dramatic change from a very viscous liquid to a highly cross-linked rigid polymer structure made of F-actin. This structure is dense enough to prevent granules from entering it. The depth of penetration of the granules and the leading edge of the pseudopod defines the apparent boundaries of the polymer. Initially, the pseuodopod extends away from the cell body. After the pseudopod extends 3 to 5 pm, granules from the cell body begin to penetrate into the pseudopod domain (Fig. 1). This defines the trailing boundary of the pseudopod. Both the extension of the polymer network and the movement of its trailing boundary proceed at a certain rate. This rate is given by the slope of the plot of the measured position vs. time of the two boundaries (see Fig. 3). It is seen from Figure 3 that both rates are constant, which suggests that the processes related to these rates-namely , the process of network formation and the process of network degradation-proceed with the same rate. The first event of fMLP induced pseudopod formation is the binding of the chemoattractant to its receptor [Posner et al. , 19941. This binding leads to the formation of a ternary complex and a release of activated G protein. Even though the cascade of events following the release of G protein is not well known, it is commonly observed that the various cellular responses depend on the concentration of the chemoattractant [Omann et al., 19871. Therefore, it is expected that the rate of network extension will be a function of fh4LP concentration in the vicinity of the pseudopod’s leading edge. Because the hydrodynamic conditions for fMLP delivery are similar for all experiments, the fMLP concentration in the vicinity of the leading edge is proportional to its concentration in the small pipet. Figure 4 shows the dependence of the measured rate of network extension on the fMLP concentration in the small pipet for three different temperatures. The dependence of the rate of network extension clearly depends on the fMLP concentration. The fMLP concentration corresponding to the rate equal to half of the measured maximum rate for a given temperature (Fig. 4) corresponds to the apparent equilibrium dissociation constant. For 30°C the value of this constant found from the data in Figure 4 is 5 x lo-’’ M. A “saturation” of the measured rate is observed at high F-Actin Network Formation 335 Fig. 2. Formation of a pseudopod-like structure in a tether. The tether is formed as the bead coated with mAb to CD18 is touched to the cell surface for 3 s and then withdrawn. Pulling a tether initiates the formation of a network. This network advances along the tether and continues to distances up to 7 to 10 km, and then it starts to retract. As the network advances it is followed by granules from the cell body. The advancing granules and the leading boundary of the network define the network region. The volume of the network region of the pseudopod like structures is small compared to the same region of fMLP stimulated pseudopods (see Figs. 1 and 10). The temperature is 22°C. fMLP concentrations. FMLP concentrations on the order of lop7 M is the “saturation” concentration for the temperatures studied. The Arrhenius plot (Fig. 5) of the rates at “saturation” is used to calculate the apparent activation energy of network formation. The apparent activation energy of network formation is found from the slope of the line approximating the experimental data in Figure 5 multiplied by the gas constant. Its value is 36.6 kJ.mol-’ (8.7 kcal-mol-’). The extent of the network during pseudopod formation always reaches a maximum. The maximum extension depends both on the fMLP concentration and the temperature. Usually large maximum extensions are measured at high temperatures. After the maximum extension is reached, two types of behavior are observed: (1) the pseudopod starts to retract immediately, or (2) the pseudopod maintains its maximum length for several minutes-in some cases up to 10 min-and then retracts. Different cells follow one or the other of the two path- ways. In these experiments more than 90% of the observed cells follow the first pathway. [It is important to note that fMLP is present in these experiments during both the extension of the pseudopod and its retraction. Modeling of the distribution of fMLP around the cell surface (Alteraifi and Zhelev, unpublished results) shows that 15 s after starting the flow of chemoattractant, its distribution becomes constant and remains unchanged (within 5%) for the next 20 to 30 min.] The rearrangement of the cytoskeleton in the pseudopod region is coupled with its contraction in the region of the main call body. Figure 6 shows the measured apparent cortical tension during fMLP-stimulated pseudopod formation. It is seen that the extension of the pseudopod is coupled with an increase in the cortical tension and its retraction with a relaxation of the cortical tension. The increase of the cortical tension usually begins after the initiation of pseudopod growth. The delay between the initiation of pseudopod growth and the increase of the cortical tension 336 Zhelev et al. s c -2.4 \ -2.8 t t ' o l = /' ' ' 0 I " ' ' ' " " 100 50 " " 150 I Fig. 3. Distance vs. time of the leading (closed circles) and trailing (open circles) boundary of the network region in a fMLP stimulated pseudopod. The boundaries move with constant velocities and the distance between them remains almost constant. The rate of advancement of the leading b o u n d q is on the order of 0.13 pms- The zero time is when the cell is exposed to fMLP at a concentration of 1 X M and the temperature is 22°C. The data are from the cell shown in Fig. 10. '. 0.25 1 0.20 ! h 7 z ' ' " 1 1 ' ' ""'"' ' """" ' """" ' """" 3.35 3.4 3.45 3.5 3.55 3.6 3.65 1000/T (K-') Fig. 5 . Arrhenius plot of the rates of network formation shown in Figure 4 at fMLP concentrations equal to the concentration of saturation. The slope of the straight line found by the least square method is -4.4-K. This slope multiplied by the gas constant R represents the apparent activation energy of network formation. The calculated apparent activation enegy of network formation is 36.6 k.l*mol-'. muth, 19951. After relaxation, the value for the cortical tension usually is larger than its initial value. '"''7 Network Formation in Tethers 0.15 3. v > 0.10 I -?', 0.00 10.ll 3.3 200 Time (s) - lo"o 10.9 10-8 , , ,,,,,, 10'7 , , , ,,,,,, , 10'6 , , ,A 10'5 WLP Concentration (M) Fig. 4. Dependence of the rate of pseudopod extension (or network formation) on the temperature and the concentration of fMLP in the small pipet (see Fig. 1). Curve 1 is for 5"C, curve 2 for 22"C, and curve 3 for 30°C. The concentrations for half of the measured maximum rate of extension for 5", 22", and 30°C are 2 X lo-', 2 X lop9, and 5 x lo-'' M, respectively. When the fMLP concentration is on M (concentration of saturation) or larger, the the order of 1 x measured rates are maximum. measured from 13 cells is 26 & 13 s. The average maximum cortical tension is on the order of 0.15 mN-m-'. The increase of the cortical tensions has a large deviation from cell to cell and most probably is related to the state of the cell cortex before stimulation [Zhelev and Hoch- Pulling a tether from the neutrophil surface initiates the formation of a pseudopod-like structure (Fig. 2). Similar to the case of an fMLP-stimulated pseudopod, the pseudopod-like structure in tethers advances along the tether at a constant rate (Fig. 7) until it reaches its maximum extension, and then it retracts. As in the case of fMLP-stimulated pseudopods, the polymer network advancing in the tether is bound between a leading and trailing boundary. The rate of advancement of the two network boundaries is similar, which provides an almost constant thickness of the network region. The temperature dependence of the process of network formation in tethers (Fig. 8) is similar to that in fMLP-stimulated pseudopods (Fig. 5). This dependence is again used to measure the apparent activation energy of network formation. This apparent activation energy is on the order of 34.9 kJ.mol-', which is similar to the apparent activation energy for the formation of fMLP-stimulated pseudopods. Site of Network Formation In the study of fMLP-stimulated pseudopod formation illustrated in Figure l , the length of the pseudopod increases from 0 to 8 pm while its thickness remains almost constant. Also, during pseudopod growth when the pipet carrying fMLP is removed and the cell is washed by moving it in the chamber, the pseudopod F-Actin Network Formation 10 “ ‘ ‘ I , , , # , , , I , , , , , , 10.20 - 2 -2.4 2 n 50 150 in0 0.00 200 Time (s) Fig. 6. Cell body contraction T, (open squares) during fMLP stimulated pseudopod growth (closed circles). The contraction of the main cell body increases as the pseudopod extends and decreases when it is about to retract. There is a delay of about 30 s between the start of pseudopod growth and the increased contraction of the main cell body. The zero time is the time when the cell is exposed to the chemoattractant and the temperature is 20°C. 12 I , , ’ ’ b s’ lo[ ” 0 1 0 0 50 100 150 200 250 300 350 Time (s) Fig. 7. Polymerization and retraction cycles for network formation in tethers. The leading (closed circles) and the trailing (open circles) boundaries of the network are shown. The temperature is 22°C. In this experiment, a single tether is pulled and the dynamics of formation of a pseudopod-like structure is measured. Initially, the network extends along the tether with almost constant velocity. When the leading boundary of the network reaches a distance from the cell body of 10 to 15 pm, its growth ceases and the pseudopod-like structure starts to “retract” (compare Fig. 2e and f ) in a process similar to pseudopod retraction shown in Figure 3. The retraction proceeds until the pseudopod-like structure disappears almost completely (Fig. 2f), and then it begins to grow all over again. This cyclic behavior is observed in almost all experiments. begins to retract (Fig. 9). This behavior suggests that network formation occurs next to the leading boundary. Then, it may be expected that proteins from the cytoplasm membrane are involved this process. If so, then , IT I # I , # , , , , , , , , l . , , , , , , 337 , , , , . ji. ~ -3.4 3.3 3.35 3.4 3.45 3.5 3.55 3.6 lOOOiT (K-’) Fig. 8. Arrhenius plot of the rates of network advancement in tethers for different temperatures. The slope is -4.2.K, which corresponds to an apparent activation energy of network formation on the order of 34.9 kl.moI-’. their “removal” from the network’s boundary is likely to affect actin polymerization. The cytoplasm membrane can be “stripped” from the leading boundary of spontaneously formed pseudopods by pulling a tether (for details see Materials and Methods) from the pseudopod’s leading boundary. [After the tether is pulled the pseudopod starts to retract (Fig. 9)]. When the tether is pulled from the leading boundary of a large lamellae, the network formation in this region ceases, while in regions far from the disturbed area it continues. The retraction of the pseudopod, after pulling a tether from its leading boundary, suggests that membrane proteins are crucial for network formation. DISCUSSION The formation of pseudopods in neutrophils can be stimulated by soluble chemoattractants [Cassimeris and Zigmond, 19901 as well as by ligands of the adhesion receptors [Lofgren et al., 1993; Brennan et al., 1991; Jaconi et al., 19911. This suggests that while pseudopod formation does not require the presence of a surface [Condeelis, 19931, the interaction of the cell with adjacent surfaces may alter its activation. To minimize the effect of the adjacent surfaces, the neutrophils used in this work are held with micropipets. This helps both to minimize the effect of adhesion as well as to produce highly oriented single pseudopods and pseudopod-like structures (Figs. 1 and 2). In the experiments on network formation in tethers, the membrane at the tip of the tether is “attached” through the binding of integrins to their antibody on the bead surface. As mentioned already, the binding of in- Zhelev et al. 338 6 "" " " ' " " " " " " ~ " " " ' " " " ' " ' ~ Average pseudopod length 0 * o 0 a tether pulled 0 0 0 10 20 30 40 50 60 70 80 Time (s) Fig. 9. Network formation in spontaneously growing pseudopods and retraction after tether formation. Two spontaneously growing pseudopods are shown (open and closed circles, respectively). The pseudopods extend linearly with time until they reach the average maximum extension shown by the dashed line. When a tether is pulled from the leading edge (arrows) of a growing pseudopod, the pseudopod starts to retract. The retraction of the pseudopod starts a few seconds after pulling the tether. tegrins to their antibodies can result in cell stimulation. To assure that receptor-antibody binding has a minimum affect on the measured data, the rate of network advancement in tethers pulled by latex beads is compared to that in tethers pulled by small pipets [Zhelev and Hochmuth, 19951. This comparison shows that for the same temperature and media, the measured velocities in both cases are similar. The possibility of neutrophil stimulation by the binding of adhesion receptors to their antibodies was also explored by measuring calcium transients in the cell cytoplasm. It is known that the crawling of neutrophils on surfaces coated with vitronectin is coupled with the occurrence of calcium transients [Marks and Maxfield, 19901. Vitronectin is a ligand for the integrin adhesion receptors, so similar behavior is expected after the binding of CD18 to its monoclonal antibody. Indeed, after keeping the cell and the bead in contact for 30 to 60 s, calcium transients were measured by measuring the fluorescence from the calcium probe Fluo-3 (Alteraifi and Zhelev, unpublished observations). However, when a tether is pulled, both by using a latex bead and by using a small pipet, calcium transients were not observed. (A total of 15 cells was observed in two different experiments.) There were several factors that may have contributed to the negligible (if any) effect of the binding of adhesion receptors at the tether tip to cell stimulation in these experiments. First, the time of cell-bead contact before pulling the tether was quite small, usually less than 3 s, compared to the time of contact when calcium transients were observed. Second, both during the initial bead-cell contact and after the tether was pulled, there were few receptors bound to antibodies. Third, these few bound receptors provide few signal molecules for cell stimulation. Fourth, the signal molecules have to "travel" the distance along the tether by either diffusing along the membrane or in the f-actin filled tether. (Zhelev and Hochmuth [ 19951 have shown that 20 to 25 s after a tether is pulled it becomes stiff, probably because of the formation of crosslinked F-actin in its interior.) Whatever the factors leading to a negligible stimulation of the cell by the attached receptors at the tether tip, they provide conditions for studying network formation in tethers pulled by latex beads instead of using small pipets. The use of latex beads significantly simplified the experimental procedure and allowed many cells in the same preparation to be studied on the same day. The pseudopods produced using fMLP-filled pipets are supported by F-actin network and are stiff, while the main cell body maintains its liquid nature. The diameter of the pseudopod region remains constant until the maximum extension is reached (Fig. 1). After that, the pseudopod diameter increases both when the pseudopod starts to retract (Fig. 1) or when it remains at maximum extension (not shown). Another feature of these pseudopods is that they have a dense network located next to the pseudopod's leading edge that excludes cell granules. The apparent thickness of this network remains constant while the pseudopod advances (Fig. 3). The same thickness for the pseudopod-like structure formed in a tether is also constant (Fig. 7). Another important observation giving an insight into network dynamics is the retraction of the pseudopod after the "stripping" of the cytoplasm membrane from its leading boundary (Fig. 9). All these observations taken together suggest that the formation of F-actin network in pseudopods and/or pseudopod-like structures occurs at the network's leading boundary (Fig. 10B). This is consistent with the hypothesis of boundary polymerization [Zhu and Skalak, 1988; Evans and Dembo, 1990; Evans, 19931. The almost constant distance between the leading and the trailing boundary of the network is consistent with the nucleation-release model [Theriot and Mitchison, 1991; Zigmond, 19931. There, new actin filaments are created near the leading edge and remain trapped in their place as the leading boundary of the network continues to advance. The trapped filaments depolymerize until eventually they disappear. The depolymerized actin is transported to the polymerization region and is partially consumed in the polymerization process. This exchange of actin between the polymerization region and the network volume is known as F-actin turnover [Zigmond, 19931. Another feature of the pseudopods studied in this work is that the overall rate of their growth is almost F-Actin Network Formation 339 -Network region Trailing boundary h <g boundarjl Network region- Pseudopod region i ZfMLP Pseudopod region I Fig. 10. a: Formation of a thick pseudopod stimulated by a pipet filled with fMLP. The penetration of granules into the pseudopod region is more clearly seen in thick pseudopods than in thin pseudopods (compare to Fig. 1). Granule penetration determines the apparent trailing edge of the F-actin network in the pseudopod region. The distance between the trailing boundary and the leading edge of the pseudopod is almost constant during the pseudopod growth. b: Schematic of the boundary polymerization model. The F-actin network is formed in the boundary polymerization region, which is next to the plasma membrane. The amount of polymerized actin is proportional to the number of occupied chemoattractant receptors in this region. During pseudopod extension, the formed F-actin filaments remain trapped in the network while the polymerization region continues to advance. The trapped filaments depolymerize until they eventually disappear. Because F-actin filaments have different initial lengths and because they are cross linked with different cross linking proteins, some of the filaments depolymerize faster than others. This provides conditions for the formation of vacancies in the network and for the penetration of granules in this region. At the same time, slowly depolymerizing filaments provide a support for the pseudopod shape. The overall rate of pseudopod advancement is determined by the rate of network formation. constant. Pseudopod growth consists of the movement of many “ruffles” that “appear” at the pseudopod leading edge (see Figs. 1 and 10). Some of the “ruffles” advance more rapidly than others, then eventually these “ruffles” slow down while others start to move more rapidly. This difference in the movement of the “ruffles” contributes to the roughness of the shape of the leading edge of the pseudopods observed in the experiments (see Figs. 1 and 10). However, regardless of the different movement of individual ‘‘ruffles,’ ’ the overall extension of the pseudopod region is almost constant (see Figs. 3 and 9). Also constant, or nearly so, is the advancement of the network region in pseudopod-like structures in tethers (Fig. 7). Similar to pseudopod ex- tension, the movement of the trailing boundary of the network region is almost constant (see Figs. 3 and 7). The overall linearity of both pseudopod extension and movement of the trailing boundary suggests that overall the network formation is a steady-state process and the amount of formed network per unit time equals the amount of F-actin that is depolymerized during the same time. In these experiments, the characteristic time for F-actin turnover is of the order of 20 s, which is obtained by dividing the typical 2 p,m thickness of the polymer domain next to the leading edge by the characteristic velocity of 0.1 p,m.s-’. The processes involved in network polymerization include: transport of actin to the site of polymerization, the dissociation of actin monomers 340 Zhelev et al. from their sequestering proteins, the formation of new nucleation sites, and F-actin polymerization. The first process has a physical nature, while the other three processes depend mainly on the rate of various chemical reactions. Whether the rate-limiting process for network formation has either a physical or chemical nature is discussed below. The physical process that may limit the rate of network formation is the transport of actin to the site of polymerization. Because the rate of network formation is constant and there is an F-actin turnover, this transport can be diffusion limited. The diffusion in this case is a steady state diffusion, where the characteristic time of diffusion T~ of a given actin species to the polymerization region is calculated by where ID is the diffusion distance and D is the diffusion coefficent. The diffusing actin species that may provide a pool of actin for polymerization are G-actin and non attached F-actin filaments. The characteristic time of diffusion for these actin species is estimated in Appendix A. From the estimate it is seen that the diffusion of G-actin is much faster than the rate of pseudopod advancement, while the diffusion of F-actin probably is much slower. Therefore, it is likely that neither determines the rate of pseudopod extension, unless both combine in some unique way to produce the measured growth rates. Something other than simple diffusion must be the ratelimiting process for pseudopod extension. The data shown in Figure 4 show that the rate of pseudopod extension depends on the chemoattractant concentration in the small pipet. This dependence is similar to the binding of fMLP to its receptor [Sklar et al., 19851. The chemoattractant concentration for observing half of the maximum rate of pseudopod extension at room temperature, calculated from the data in Figure 4, is on the order of 2 X M. This value is of the order of the dissociation constant of fMLP [Tomhave et al., 19941 and FNLPNTL [Sklar et al., 19851. All these data taken together suggest that the rate of pseudopod extension is unlikely to depend on the transport of actin to the site of polymerization. This rate most probably depends on the rate of the chemical reactions triggered by the binding of the chemoattractant to its receptor. These reactions include the formation of a ternary complex [Posner et al., 19941, release of activated G protein, activation of phospholipase C , production of the secondary messengers inositol triphosphate and diacylglycerol, the activation of protein kinase C , and a sequence of not yet well-studied reactions that result in network formation [Omann et al., 1987; Devreotes and Zigmond, 1988; Snyderman and Uhing, 19921. Even though the exact chemical reactions are unknown, the fact that the rate of network formation is almost constant (Fig. 3) and that this rate depends on the chemoattractant concentration (Fig. 4) suggests that the involved reactions are steadystate reactions and are far from equilibrium. In the boundary polymerization approximation used here (see Fig. lOB), it is assumed that actin polymerizes at the boundary (or the region) between the network and the membrane. As the polymerization proceeds, the boundary is displaced from its previous position and depolymerization becomes the dominant feature of the network, away from the boundary. Because of the movement of the boundary the chemical reaction in the boundary polymerization region is far from equilibrium, while at the same time it proceeds at a constant overall rate. These characteristics suggest that network formation can be modeled as an apparent “enzymne” reaction (see Appendix B). The calculated apparent Gibbs energy of this reaction is on the order of 43 kJ.mol-’ (10 kcal.mol-’), which is on the order of the Gibbs energy for ATP hydrolysis. The apparent Gibbs energy does not represent the true Gibbs energy of the rate limiting reaction because it is not known how the rate constant k, is related to the rate constants of the involved chemical reactions. Even though the rate limiting reaction of network formation is unknown, the comparison of the Arrhenius plots of network formation in fMLP stimulated pseudopods and in tethers shows that the apparent activation energy on both cases is similar. This similarity suggests that both the process of chemoattractant-stimulated network formation and the process of network formation in the absence of chemoattractants share the same rate limiting reaction. Pseuodpod formation stimulated by fMLP is coupled to an increase in the cortical tension of the main cell body (Fig. 6). Because the neutrophil cortex forms an apparent “shell” next to the cell surface [Zhelev et al., 19941, the increase of the cortical tension leads to an increase of the static pressure in the cell cytoplasm. It has been suggested [Stossel, 1993, see also the reviews of Codneelis, 1992, 19931 that the increase of the cytoplasm static pressure may be the driving force for cytoplasm flow and pseudopod extension. In previous work on yeast phagocytosis [Evans et al., 19931 and mechanically stimulated cortical rearrangement [Zhelev and Hochmuth, 19951, the increase of the cortical tension was delayed until about 30 s after the initiation of cortical rearrangement and cytoplasmic flow. This delay is also observed here (see Fig. 6), where the delay between initiation of pseudopod growth and cell body contraction is on the order of 26 s. Therefore, the contraction of the main cell body may account for the cytoplasm flow and F-Actin Network Formation 341 cell shape changes only at the final stages of cortical Cassirneris, L., and Zigmond, S. H. (1990): Chemoattractant stimulation of polymorphonuclear leukocyte locomotion. Semin. rearrangement. This observation is in accord with other Cell Biol. 1:125-134. biochemical studies [Condeelis, 19921 showing that it is Cassirneris, L., McNeill, H., and Zigmond, S. H. (1990): Chemoatunlikely for the cell body contraction to be the major tractant-stimulated polymorphonuclear leukocytes contain two driving force of pseudopod extension. Cell body contracpopulations of actin filaments that differ in their spatial distributions and relative stab es. J. Cell Biol. 110:1067-1075. tion and the related force generation are most probably Condeelis, J. (1992): Are all pseudopods created equal? Cell Motil. involved in the later stages of cell motility. Cytoskeleton 22:l-6. In conclusion, the results of this work are consisCondeelis, J. (1993): Life at the leading edge: The formation of cell tent with the proposed boundary polymerization model, protrusions. Annu. Rev. Cell Biol. 9:411-444. where actin polymerizes at the boundary region between Devreotes, P. N., and Zigmond, S. H. (1988): Chemotaxis in eukarotic cells: A focus on leukocytes and Dictyostelium. Ann. the network and the cytoplasm membrane. As the polyRev. Cell Biol. 4549-686. merization proceeds the formed filaments are trapped in Evans, E. A. (1993): New physical concepts for cell amoeboid rnothe network where they depolymerize. For a completely tion. Biophys. J. 64:1306-1322. extended pseudopod the polymerization at the boundary Evans, E., and Dembo, M. (1990): Physical model for phagocyte and the depolymerization in the network volume are balmotility: Local growth of a contractile network from a passive body. In Akkas, N. (ed.): “Biomechanics of Active Movement anced in a way that keeps the amount of polymer in the and Deformation of Cells.” NATO AS1 Series, Vol. H42. pseudopod constant [Zigmond, 19931. The overall rate Berlin: Springer-Verlag, pp. 185-214. of network formation is limited by the rate of the inEvans, E. A., and Yeung, A. (1989): Apparent viscosity and cortical volved chemical reactions, where an enzyme type of tension of blood granulocytes determined by micropipet aspireaction is the rate-limiting one, and the rate of network ration. Biophys. J. 56:151-160. formation is proportional to the concentration of occu- Evans, E., Lung, A., and Zhelev, D. (1993): Synchrony of cell spreading and contraction force as phagocytes engulf large pied N-formyl peptide receptors in the membrane. The pathogens. J. Cell Biol. 122:1295-1300. polymerization reaction is endothermic (Appendix B) with an apparent Gibbs energy at 37°C on the order of 10 Felder, S., and Kam, 2. (1994): Human neutrophil motility: Timedependent three-dimensional shape and granule diffusion. Cell kcal-mol- . Motil. Cytoskeleton 28:285-302. ’ ACKNOWLEDGMENTS This work was supported by a grant from the Whitaker Foundation to D.V.Z., a predoctoral fellowship from the United Arab Emirates University to A.M.A., and a grant 5 R01 HL23728 from the National Institutes of Health to R.M.H. Blood drawing was supported by grant MO 1-RR-30 from the National Institutes of Health to the Rankin Clinical Research Unit of Duke University. REFERENCES Berland, K. M., So, P. T. C., and Gratton, E. (1995): Two-photon fluorescence correlation spectroscopy: Method and application to the intracellular environment. Biophys. J. 68:694-701. Bicknese, S., Periasamy, N., Shobet, S. B., and Verkman, A. S. (1993): Cytoplasmic viscosity near the cell plasma membrane: Measurement by evanescent field frequency-domain microfluorimetry. Biophys. J. 65:1272-1282. Brennan, P. J., Zigmond, S. H., Schreiber, A. D., Smith, E. R., and Southwick, F. S. 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Cell Biol. 1121249-1257. Kao, H. P., Abney, J. R., and Verkman, A. S. (1993): Determinants of the translational mobility of a small solute in cell cytoplasm. J. Cell Biol. 120:175-184. Lanni, F., and Ware, B. R. (1984): Detection and characterization of actin monomers, oligomers, and filaments in solution by measurements of fluorescence photobleaching recovery. Biophys. J. 4697-110. Lawson, M. A., and Maxfield, F. R. (1995): Ca2+-and calcineurindependent recycling of an integrin to the front of migrating neutrophils. Nature 377:75-79. Lofgen, R., Ng-Sikorski, J., Sjolander, A., and Andersson, T. (1993): p2 Integrin engagement triggers actin polymerization and phosphatidylinositol triphosphate formation in non-adherent human neutrophils. J. Cell Biol. 123:1597-1605. Luby-Phelps, K., Mujumdar, S., Mujumdar, R. B., Ernst, L. A., Gdbraith, W., and Wagooner, A. S. (1993): A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65:236-242. 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Biochemistry 21:3666-3674. Theriot, J. A., and Mitchison, T. J. (1991): Actin microfilament dynamics in locomoting cells. Nature 352: 126-13 1. Tomhave, B. D., Richardson, R. M., Didsbury, J. R., Menard, L., Snyderman, R., and Ali, H. (1994): Cross-desensitization of receptors for peptide chemoattractants. J. Immunol. 153:32673275. Watts, R. G., and Howard, T. H. (1993): Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes. Blood 10:2750-2757. Zhelev, D. V., and Hochmuth, R. M. (1995): Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils. Biophys. J. 68:2004-2014. Zhelev, D. V., Needham, D., and Hochmuth, R. M. (1994): Role of the membrane cortex in neutrophil deformation in small pipets. Biophys. J. 67:696-705. Zhu, C., and Skalak, R. (1988): A continuum model of protrusion of pseudopod in leukocytes. Biophys. J. 54:1115-1137. Zigmond, S. H. (1993): Recent quantitative studies of actin filament turnover during cell locomotion. Cell Motil. Cytoskeleton 25: 309 -3 16. APPENDIX A Estimating the Characteristic Time of Diffusion The characteristic time of diffusion of G-actin and F-actin is calculated from Eq. 2. The diffusion distance is on the order of the distance between the leading boundary and the trailing boundary of the network. This distance measured from the data in Figures 3 and 7 is of the order of 1 to 3.5 pm. The diffusion coefficient in Eq. 2 is an apparent parameter. It depends on the cytoplasm viscosity, network density, and the size of the diffusing actin species. There are two actin species that may contribute to network formation--(;-actin and F-actin. Among them G-actin diffuses faster and gives the lower bound of the characteristic time of diffusion. The diffusion coefficient of G-actin has been measured in water and is on the order of 7.1 x lo-” m2*s-l [Tait and Frieden, 1982; Lanni and Ware, 19841. The value of the diffusion coefficient when G-actin diffuses in the highly viscous cytoplasm of the neutrophil is estimated below. Measurements of the diffusion of fluorescent molecules and small beads in the cytoplasm of fibroblasts and kidney cells show that these small molecules diffuse up to five time slower than they do in water [Bicknese et al., 1993; Luby-Phelps et al., 1993; Kao et al., 1993; Berland et al., 19951. This slowing of the diffusion of small molecules is a result of volume occlusion due to the presence of the cell cytoskeleton [Kao et al., 19931. Felder and Kam  measured the diffusion of the fluorescent probe BCECF in the cytoplasm of activated human neutrophils and obtained a value of 4.3 X m2*sP1[Felder and Kam, 19941. This value is 50 times smaller than the diffusion coefficient of the same probe in water and corresponds to more than 50% volume occlusion in the neutrophil cytoplasm [Kao et al., 19931. The volume occlusion in the pseudopod region is difficult to measure because of F-actin turnover and the resulting gradient in network density. It can be estimated from electron micrographs of triton X-100-extracted residues of human neutrophils [Stossel, 19931. From these micrographs it is seen that the volume occlusion is less than 50%. Therefore, the measured decrease of 50 times in the BCECF diffusion in neutrophils by Felder and Kam [19941 may give an overestimate of the value of the diffusion coefficient for G-actin. In any case, the actual value for the G-actin diffusion coefficient may be from five [Bicknese et al., 1993; Luby-Phelps et al., 1993; Kao et al., 1993; Berland et al., 19951 to fifty [Felder and Kam, 19941 times smaller than the diffusion coefficient in water. However, because BCECF is similar in size to G actin-I nm vs. 4 nm, respectively-the diffusion coefficient for G-actin is expected to be of the same order of magnitude. Therefore, the minimum and maximum apparent diffusion coefficients are on the order of 1.4 X lo-” m2-s-’ and 1.4 x m2*s-l, respectively. Then, the time of diffusion across a 2 km dense network at the leading edge of the pseudopod region is on the order of 0.4 to 4 s. The later time is smaller than the time of F-actin turnover, which is on the order of 10 to 20s in our experiments and these of others [Cassimeris et al., 1990; Cano et al., 19911. It is seen that the time of diffusion of G-actin is smaller than the time of F-actin turnover and, thus, diffusion of G-actin is not the rate-limiting process in network formation. However, if F-Actin Network Formation non attached F-actin filaments [Cano et al., 19911 provide a significant pool of actin for the polymerization process, the characteristic time of actin diffusion can be much slower [Simon et al., 19881 than that estimated for G-actin. In this case (when non attached F-actin filaments rather than G-actin monomers provide a pool for actin polymerization), actin diffusion could be a ratelimiting process in network formation. APPENDIX B Model for Network Formation in Pseudopods Network dynamics in the pseudopod region is illustrated in Figure 10. The network filaments are formed at the membrane boundary and depolymerize as this boundary moves away. The rate limiting process for boundary movement (or pseudopod extension) is the slowest chemical reaction involved in network formation. Except for the slowest reaction, all other reactions are at steady state. Also, just as it is for enzyme reactions, the amount of formed network [F] per unit time is proportional to the concentration of at least one of the chemical species involved in the slowest reaction. Therefore, because all reactions except the slowest one are steady-state reactions, the concentration of this chemical species is proportional to the number of occupied fMLP receptors [E,] (or to the bulk concentration of fMPL as shown in Fig. 3). Then, the apparent rate of network formation is (AII. 1) where k, is an apparent rate constant. The apparent rate k, can be estimated when the rate d[FI of network formation -and the number of occupied dt receptors [E,] are known. Unfortunately, the rate of network formation has not been measured in living cells, and its value must be estimated. Instead of its actual value, here its estimate is used. This estimate is found from the amount of F-actin formed after the exposure of the cells to a chemoattractant. In the experiments of Can0 et al. , neutrophils are exposed for 90 s to 0.02 p M fNLLP. After that time the amount of F-actin has doubled relative to its amount in resting cells. The total amount of actin in resting cells is about 100 pM and from this only 30% is F-actin [Southwick et al., 19891. Thus, the amount of freshly polymerized actin is on the order of 30 pM. It is assumed that most of the polymerized actin remains in the pseudopod region and is exchanged during F-actin turnover. The time of F-actin turnover is of the order of 20 s (Fig. 3). This gives for the 343 rate of network formation a value on the order of 1 pM.s-'. The rate of network formation can be converted to the number of molecules added to the network per second. This conversion is done by multiplying the above rate by the cell volume and Avogadro's number. The radius of a resting neutrophil is 4 pm. This gives for liters. Then, the rate of the cell volume 2.7 X network formation calculated above converts to 1.6 x lo5 s-l (molecules per second). The other parameter, which is needed to calculate k,, is the number of occupied N-formyl peptide receptors. At saturation, this number is equal to the number of receptors in the leading edge of the pseudopod. The number of N-formyl peptide receptors in the leading edge is calculated by taking the fraction of the total amount of receptors that reside in this region. This fraction is calculated from the ratio of the area of this region to the total cell area. The diameter of the boundary polymerization region is of the order of 3 pm (see Fig. l), which has a correspondingarea of 7 pm2. The area of the neutrophil is of the order of 200 pm'. Then, the area of the boundary polymerization region is 3.5% of the total cell area. This is the fraction of N-formyl peptide receptors residing in the pseudopod's leading edge. The total number of receptors on the membrane of a resting cell is 20,000 [Norgauer et al., 19911. Within the first 2 min after cell activation, this number increases to reach a total of 40,000 receptors, and then for the next several minutes remains almost constant [Norgauer et al., 19911. Considering the total number of receptors equal to 40,000, the number of receptors in the polymerization region is on the order of 1,400. [In this calculation it is assumed that N-fonnyl peptide receptors do not aggregate. This assumption is supported by the results from our recent studies, where we characterize the binding of fluorescent labeled ligands to the N-formyl peptide receptor during chemoattractantinduced pseudopod formation (Alteraifi and Zhelev, unpublished results)]. Using the value of 1.6 X lo5 s-' for the rate of network formation and the value of 1,400 for the number of N-formyl peptide receptors in the boundary polymerization region, the calculated apparent value of k, is 1.1 X lo2 s-'. The estimated value of k, is used to calculate the + apparent Gibbs energy of network formation AG+ [Glasstone et al., 19411. This is done by using + + f AG+ = AH+ - TAS+ + (AII.2) + where AH+ and AS+ are apparent enthalpy and entropy, respectively, and T is the absolute temperature. The en- + thalpy is calculated from: AH+ = E, - RT, where E, 344 Zhelev et al. is the activation energy of network formation given by the slope of the Arrhenius plot in Figure 5 , and R is the gas constant. The entropy is calculated from: + =Rin( g) 9 (z). where h is the plank constant, and the / +\ parameter Y is: Y = k,exp - The apparent en- thalpy and entropy are 34 kl-mol-' (8 kcal-mol-') and -30 J.mol-'.K-' (-7 cal-mol-'-K-'). The calculated Gibbs energy for 37°C is on the order of 43 kJ-mol-' (10 kcal-molThe apparent enthalpy is positive, which indicates that the process of network formation is endothermic. '>.