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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Department of Biomedical Engineering, Washington University, Saint Louis, Missouri
Submitted 8 May 2006 ; accepted in final form 24 October 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50 pN and the effective viscosities are around 0.5 pN·s/µm. These results, which are close to what was obtained from HUVECs, indicate that homogeneity is preserved in terms of tether extraction among different types of endothelial cells, and simultaneous tethers are likely extracted when leukocytes roll on either microvascular or macrovascular surfaces. leukocyte rolling; cell mechanics; micropipette; cytoskeleton
eff). F0 is the minimum force required to extract a tether from the membrane, which is determined by the membrane tension, the membrane bending stiffness, and the membrane-cytoskeleton association energy, whereas eff is determined by the membrane viscosity, the interbilayer slip, and the membrane slip over the cytoskeleton (10). From the study of single-tether extraction from lipid vesicles and several types of cells, including erythrocytes, neuronal growth cones, and neutrophils, it has been shown that (6, 10, 26)
 | (1) |
eff is 1.8 pN·s/µm for human neutrophils. Since leukocyte rolling is a dynamic process correlated with both leukocytes and endothelial cells, for a better understanding of this process, it is also essential to investigate the property of tether extraction from endothelial cells. Tether extraction from HUVECs has already been conducted using the MAT, and the same linear relationship shown in Eq. 1 was found (8). For HUVECs, the effective viscosity of 0.5 pN·s/µm was strikingly smaller (approximately threefold) than that of passive neutrophil, but the threshold force of 50 pN was similar, which enables simultaneous tether extraction from both leukocytes and endothelial cells. According to a simple mechanical equilibrium model, simultaneous tethers should further stabilize leukocyte rolling on the endothelium compared with tether extraction from either cell alone (8). However, rolling of leukocytes on endothelial cells occurs primarily in the microvasculature, and accumulating evidence has shown that there are considerable differences between endothelial cells derived from macrovascular vessels and those from microvascular vessels (13). In response to inflammation, these cells differ because of their distinct lineages, notably in the expression and regulation of cell adhesion molecules and the response to cytokine stimulation; yet whether they have different tether mechanics is not known.
In addition to cell membrane, the cytoskeleton, especially the actin-filament network, also plays an important role in the progress of tether extraction (10). Both the threshold force and effective viscosity are largely determined by the mechanical property of the cytoskeleton and the association between the cytoskeleton and cell membrane. Although the contribution of the cytoskeleton to tether extraction from human neutrophils has already been studied by treating the cells with actin-disrupting reagents (14, 28), the role of actin filaments in tether extraction from endothelial cells remains unclear.
In this paper, we studied tether extraction from two types of microvascular endothelial cells (MECs) using the MAT: adult and neonatal dermal MECs. Membrane receptors were used as handles for imposing pulling forces, and antibody-coated latex beads were used as the force transducer of the MAT. The effects of different receptor types, cell attachment states, and cytokine stimulation were studied, and the results were compared with that of HUVECs. The role of actin filaments in tether extraction was evaluated by disrupting F-actin with latrunculin A. We found that tether extraction from MECs was extremely similar to tether extraction from HUVECs from a mechanical viewpoint. Therefore, as predicted for HUVECs, tethers are very likely extracted from MECs when leukocytes roll in microvascular vessels.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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For cytokine stimulation of endothelial cells, 10 ng/ml tumor necrosis factor-
(TNF-; R&D Systems, Minneapolis, MN) and 10 ng/ml interleukin-1
(IL-1; also from R&D) were applied to HDMECs-a and HDMECs-n, respectively. The reagents were added 6 h before the experiments to induce maximal expression of cell adhesion molecules (13). TNF- of 10 ng/ml has been found to have no cytotoxic effect on the endothelial cell (15). To study the effect of actin cytoskeleton on tether extraction, we treated endothelial cells with latrunculin A to disrupt the actin filaments. Latrunculin A was purchased from Calbiochem (La Jolla, CA) and prepared as 2 mM stock solution in DMSO. The stock solution was stored at –20°C and diluted immediately before the experiments. The final concentration of latrunculin A in the experimental chamber was 5 µM, and the experiments were performed after the cells had been incubated for 20 min at room temperature.
Bead preparation.
Spherical latex beads were employed as the force transducer of the MAT. Three drops of latex beads (8 µm in diameter, Sigma, St. Louis, MO) coated with goat anti-mouse IgG were washed with 1 ml PBS twice and then coated with mouse anti-human antibodies (5 µg/ml) at 37°C for 1 h after being resuspended in 1 ml PBS. Four different mouse anti-human mAbs were used in the experiments: anti-CD31, anti-CD54 (R&D Systems), anti-CD29, and anti-CD62E (BD Pharmingen, San Diego, CA). After the incubation with antibody, the beads were kept in PBS at 4°C. Before use, 20-µl beads were taken out and washed with PBS twice.
Tether extraction.
The micropipette preparation and manipulation were described in detail previously (26, 28). Briefly, an 8-µm micropipette was made to hold the force transducer in each experiment (Fig. 1). The first 10 mm of the micropipette was filled with 1% BSA, and the rest of it was backfilled with PBS. For the experiments with suspended cells, another micropipette was set up on the right to hold the cell with a small aspiration pressure. The pressure was so small that the cell did not deform much. In the left micropipette, an antibody-coated bead that had almost the same diameter as the micropipette was aspirated into it. With different aspiration pressures, the bead could move freely with different velocities in the pipette. In a typical contact event, an expelling pressure was first superimposed on the suction pressure to drive the bead toward the cell. After the bead briefly touched the cell, the expelling pressure was released and the bead either adhered or immediately moved away from the cell under the remaining suction pressure. When the suction pressure was large enough, the adherent bead moved downstream with a tether extracted from the cell. In this case, the bead velocity, which was equal to the tether growth velocity, was clearly smaller than the velocity of a freely moving bead under the same suction pressure. Such a motion pattern is shown by the open circles in Fig. 2. With different suction pressures, tethers with different velocities could be drawn from the cell membrane. The magnitude of the force imposed on the spherical transducer can be calculated with (26)
 | (2) |
p is the suction pressure, Rp is the radius of the micropipette, and Uf is the bead velocity when it is moving freely under p. 
= (Rp – Rs)/Rp, where Rs is the radius of the bead. All these experiments were performed at room temperature.
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30 nm (28). The diameters of the left pipette and bead were determined with the DIC and bright-field microscopy, respectively, and divided by their corresponding correction factors (28). Fluorescence microscopy. To visualize the actin filament, attached cells grown on coverslips were fixed in 3.7% formaldehyde for 30 min at room temperature and then rinsed with PBS three times. Fixed cells were then stained with Alexa Fluor phalloidin (Molecular Probes, Eugene, OR) for 1 h and washed extensively with PBS three times to eliminate background noise. For nonadherent cells, the cell suspension was incubated in a centrifuge tube, first with the fixative and then with the Alexa Fluor phalloidin with the same concentration and duration as in treating the attached cells. After each incubation, the cells were washed three times with PBS by centrifugation and suspended in PBS for direct observation. The effect of latrunculin A on the cells was examined by treating the cells with 5 µM latrunculin A for 20 min before the fixation and observation.
Statistical analysis. The slopes and intercepts between two linear regression lines were compared with two-tailed Student's t-test (36). Among multiple regression lines, the comparison of the slopes was performed with ANOVA, and the comparison of the intercepts was performed with Tukey's test as described by Zar (36). The significance level was chosen to be 0.05 for all the comparisons.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-integrin) are constitutively expressed on unstimulated MECs, whereas the expression level of CD54 (intercellular adhesion molecule-1) is comparatively lower. However, after 6 h of stimulation with either TNF- or IL-1, the expression of CD54 is upregulated and the expression of CD62E (E-selectin) is also upregulated.
To examine whether tethers could be extracted from HDMECs-a, we exerted point-pulling forces between 50 and 150 pN on the membrane surfaces of these cells. The contact time and contact area between the bead and cell were chosen to achieve an adhesion frequency of <20% so that only one tether was extracted in most of the adhesion events (25). The adhesion frequency is determined by dividing the total number of adhesion events by the total number of contacts. In addition, single-tether extraction was inferred from the pattern of bead motion. As shown in Fig. 2, the bead accelerated only once before it reached its free motion velocity, whereas more than one acceleration were observed in the bead motion for double or multiple tether extraction (7). Shown in Fig. 3 is the correlation between the pulling force and the tether growth velocity for single-tether extraction obtained from unstimulated suspended HDMECs-a. For this specific case, the beads were coated with anti-CD31 antibody. A total of 223 tethers was pulled out from 23 cells. Figure 3 shows that single tethers could indeed be extracted from suspended HDMECs-a using CD31 as a force handle, and the relationship between tether growth velocity and pulling force can be described by Eq. 1 very well. Fitting the data in Fig. 3 with Eq. 1 yielded a threshold force (intercept) of 50 pN and an effective viscosity (slope divided by 2
) of 0.48 pN·s/µm, which are close to what was obtained with HUVECs (8).
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-stimulated endothelial cells (both suspended and attached) was also studied. We used three different antibodies (anti-CD31, anti-CD29, and anti-CD54) in the experiments with unstimulated endothelial cells (suspended or attached) and two different antibodies (anti-CD54 and anti-CD62E) in the experiments with stimulated endothelial cells (suspended or attached). For these 10 groups of cells studied, a total of 1,564 tethers was extracted from 191 cells. For each group, the pulling force is linearly dependent on the tether growth velocity as shown in Eq. 1. A summary of all the threshold forces and effective viscosities, as well as their associated 68% confidence intervals, is shown in Table 1. It is obvious that, for all the cases, the threshold forces and effective viscosities are close to each other (Fig. 4) and similar to what was obtained with HUVECs (8). Consequently, we can hypothesize that the cell attachment state (suspended or attached), stimulation, and receptor type have no effects on tether extraction from HDMECs-a. To examine this hypothesis, we performed multiple comparisons between slopes and intercepts among these 10 regression groups. No significant difference was found among the slopes (P > 0.1), which indicate that the receptor type, attachment state, and TNF- stimulation have no impact on the effective viscosity of tether extraction from HDMECs-a. Further statistical analysis showed that there was no significant difference for the intercepts among the 10 groups either (P > 0.05). Therefore, we can conclude that the threshold force for tether extraction from HDMECs-a is independent of the receptor type, attachment state, or TNF- stimulation. For all 10 groups, the threshold force is 58 pN (SD 6) and the effective viscosity is 0.57 pN·s/µm (SD 0.07).
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. Other experimental conditions were the same as in tether extraction from HDMECs-a. The threshold forces and effective viscosities for the 10 cell groups here were summarized in Table 2, and all the data points were plotted in Fig. 5. As in the case of HDMECs-a, no statistically significant differences were found for the threshold force or effective viscosity among different groups (P > 0.05 for both parameters), which shows that tether extraction from HDMECs-n is also independent of the receptor type, attachment state, or IL-1 stimulation. For all groups, the threshold force for HDMEC-n is 55 pN (SD 4) and the average effective viscosity is 0.51 pN·s/µm (SD 0.04).
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Latrunculin A affects tether extraction from both suspended and attached endothelial cells. The cytoskeleton, especially the actin filament network, plays a critical role in tether extraction (14, 28). To evaluate how actin filaments affect tether extraction from endothelial cells, we first examined both suspended and attached HDMECs-a by fluorescence microscopy. For normal suspended cells, although they were dissociated from the substrate, a bright ring was visible at the cell periphery after the cells were stained with Alexa Fluor phalloidin (Fig. 6A), which indicates that actin filaments are densely accumulated in this area. After the treatment with 5 µM latrunculin A for 20 min, most of the bright ring disappeared (Fig. 6B), which reveals the disruption of actin filaments. In addition, treated suspended cells were softer and more flaccid compared with normal suspended cells, although almost all of them appeared to be spherical. Figure 6, C and D, shows two typical attached cells before and after the treatment with 5 µM latrunculin A for 20 min. It is clear that, after the treatment, the stiff and straight actin filament bundles (or stress fibers) were completely disrupted. Moreover, the treated attached cells shrank from the periphery and their thickness increased, which could be seen when they were observed on the side wall of the experimental chamber as in Fig. 1B.
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0.05 pN·s/µm for both suspended and attached cells, and the threshold forces decreased to 30 pN, both of which are significantly less than the values obtained in tether extraction from native cells with the same membrane receptor (P < 0.01 for both parameters and cell attachment state).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We found a linear relationship between the pulling force and tether growth velocity (Eq. 1) for tether extraction from endothelial cells. In addition to this linear relationship, a power law relationship has also been proposed (9). This power law relationship exhibits a shear-thinning behavior, and it was fitted very well to the data obtained from neutrophil tether extraction over a larger range of tether growth velocities from 0.4 to 150 µm/s. A recent model based on the assumption of tether narrowing during extraction also suggests this power law relationship (2). However, in the present study, the tether growth velocity mainly falls in the range of 3–25 µm/s, which is close to the previous finding that neutrophils generally roll in a velocity range of 10–40 µm/s under postcapillary venular wall shear stress levels in vitro and in vivo (12). In this small range, the linear relationship shown in Eq. 1 can still characterize our results very well.
Tether extraction mechanics are mainly dependent on the mechanical properties of the cytoskeleton and membrane. For example, the threshold force is mainly determined by the adhesion energy between the membrane and the underlying cytoskeleton, whereas the effective viscosity is largely dependent on the membrane slip over the cytoskeleton (10). The comparable threshold forces obtained from suspended and attached MECs indicate that endothelial cells have similar membrane-cytoskeleton adhesion energy no matter whether they are suspended or attached. If we assume the bending stiffness (B) of cell membrane is 0.2 pN·µm, the membrane-cytoskeleton adhesion energies for HDMECs-a and HDMECs-n are F02/82B 
213 pN/µm and 192 pN/µm, respectively. On the other hand, the comparable effective viscosities obtained from these cells suggest that they also have similar viscous slip between the membrane and cytoskeleton, since the membrane interbilayer slip only has a minor contribution to the effective viscosity (10).
The existence of actin filaments in suspended endothelial cells was examined with phalloidin, which only stains F-actin and does not bind to G-actin. Since no long and straight stress fibers were observed inside the cells, the bright actin-rich ring at the periphery of the cells proves the accumulation of actin underneath the membrane. This bright ring could be disrupted by treating the cells with latrunculin A, which forms a 1:1 molar complex with G-actin (4) and disrupts actin filament formation; hence the disappearance of the ring after the treatment with latrunculin A further proves that the ring is mainly composed of actin filaments. After the cells were treated with latrunculin A, the significant decrease in both the effective viscosity and threshold force demonstrates the critical role of the actin-based cytoskeleton in tether extraction. For both suspended and attached HDMECs-a, the adhesion energy between the membrane and cytoskeleton decreased by 60% after the treatment with 5 µM latrunculin A, whereas the effective viscosity decreased more than 88%. This large decrease in the effective viscosity indicates that the membrane slip over the cytoskeleton is the major component of the energy dissipation during tether extraction and that the contribution of the interbilayer slip is <12%. Similar trends were also observed in tether extraction from neutrophils after the cells were treated with 100 µM cytochalasin D (28) or 0.3 µM latrunculin A (14). For both suspended and attached HDMECs-a treated with 5 µM latrunculin A, the effective viscosity was around 0.05 pN·s/µm. As measured by Evans and Yeung (6, 34), the effective viscosities for vesicles such as sphingomyelin/cholesterol and 1-stearoyl-2-oleoly-sn-glycero-3-phosphocholine are 0.071 and 0.009 pN·s/µm, respectively. Therefore, the values obtained from the treated MECs are in the same neighborhood as those obtained from the lipid vesicles.
The visible membrane tubes extracted from latrunculin-A-treated cells showed that tethers did indeed exist between the bead and cell. In general, the tether radius was only tens of nanometers and it is difficult to visualize a tether with light microscopy. The tether radius is related to the adhesion energy by 
, where T is the membrane tension and 
t is the adhesion energy per unit area (10). Once actin filaments are disrupted, the adhesion energy is essentially vanished and the tether radius is therefore larger. In addition, we noticed that tethers grew much longer after the treatment with latrunculin A. This could be due to two reasons: 1) under similar pulling forces, tethers from treated cells grew much faster; and 2) the adhesion lifetime between the cell and bead became longer because of the formation of multiple bonds, which became possible after the cell membrane was flattened by the disruption of the cytoskeleton and more receptors were available for bond formation.
Under physiological conditions, endothelial cells in vivo are tightly connected to each other and to the underlying cells, so they are probably in a state that is between suspended and attached. As shown in Fig. 6, A and C, suspended and attached cells have quite different configurations of the cytoskeleton. However, the cytoskeletal component that is the most important in tether extraction is the part that is directly underneath the cell membrane or the so-called cortical cytoskeleton, as shown in Fig. 6A for suspended cells. For attached endothelial cells, only long straight stress fibers could be observed by fluorescence microscopy (Fig. 6C), but the existence of the cortical cytoskeleton has already been verified with atomic force microscopy (19). The similarity in tether extraction mechanics for both suspended and attached cells indicates that the mechanical properties of the interaction between the cytoskeleton and cell membrane are similar for the cells at different attachment states. Consequently, the threshold force and effective viscosity obtained in this study may well be applicable to the cells in vivo since attached cultured cells are also connected to the underlying substrate. They may not be tightly connected to each other, but their much-stretched morphology implies that they may be under even more tension than the cells in vivo. Whether the cells in vivo have enough membrane storage to support tether extraction is not clear. However, in a separate study of double tether extraction from endothelial cells, we found that both the effective viscosity and threshold force were doubled, which indicates that endothelial cells probably have enough membrane materials to support double tether extraction and that there is no competition between double tethers from endothelial cells (7).
Tether extraction from MECs is independent of cytokine stimulation. The effectiveness of cytokine stimulation was demonstrated by flow cytometry (data not shown). In addition, it was demonstrated by measuring the adhesion frequency between anti-CD62E-coated beads and MECs. Few adhesion events were observed between anti-CD62E-coated beads and unstimulated MECs (both suspended and attached). However, after MECs were stimulated with either TNF- or IL-1, the adhesion frequency measured from both suspended and attached cells was greatly increased, which indicates that the cells were effectively stimulated. After the cells were stimulated, no significant difference in the effective viscosity or threshold force was detected. This is contrary to what we observed in our previous studies of human neutrophils (28, 32). For neutrophils, both the effective viscosity and threshold force were modulated by the stimulation with either phorbol myristate acetate or IL-8. It has been postulated that both phorbol myristate acetate and IL-8 could cause a decrease in the membrane slip over the cytoskeleton but an increase in the membrane-cytoskeleton adhesion energy of neutrophils. Nevertheless, no such effect was observed for the MECs stimulated with either TNF- or IL-1. Although the treatment with TNF- was found to increase both the number and thickness of stress fibers in human aortic endothelial cells (29), it probably does not have any effect on the cortical cytoskeleton or its interaction with cell membrane.
Tether extraction from MECs is also independent of cell passage number. In our experiments, only cells in passages 3–5 were studied. An earlier study with the MAT found that older bovine aortic endothelial cells were stiffer than younger cells and that they also had a higher concentration of stress fibers (21). However, no significant difference was found in either the threshold force or effective viscosity obtained from the cells with different passage numbers (data not shown). In addition, no significant difference was found in tether behavior between neonatal and adult MECs, even though the two cell lines were isolated from subjects of different ages and different anatomical sites. This conclusion may indicate that the interaction between the cytoskeleton and cell membrane is fully developed in the neonatal MECs, although they may be different from the adult MECs in other aspects.
All the experiments presented in this study were performed on the cells without exposure to shear stress. However, endothelial cells in vivo are constantly subjected to blood flow and shear stress, which may affect their morphological and mechanical properties (17, 22, 23). Therefore, it is essential to investigate tether extraction from endothelial cells exposed to shear stress. As a first step, we only studied tether extraction from the MECs under static conditions and compared the results to our previous ones obtained from HUVECs. In the future, by combining the MAT and flow chamber assay, we can investigate tether extraction from endothelial cells under more physiological conditions.
Both types of MECs used in this study are derived from the microvasculature but different anatomic tissues, whereas HUVECs are isolated from large veins (8). It has been shown that differences exist not only between endothelial cells from large and small vessels (11, 31) but also between endothelial cells from vessels of the same size but different tissues (5, 16). However, as shown here, all three types of cells share the same tether mechanical properties. Compared with tether extraction from neutrophils, the similar threshold force and the significantly smaller effective viscosity indicate that more membrane will flow out from the endothelial cell when tethers are extracted simultaneously from both the neutrophil and endothelial cell during the inflammatory response. Therefore, the stabilization of leukocyte rolling by tether extraction should be attributed more to the endothelial cell than to the neutrophil.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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