Lymphocyte Mechanical Response by Cross-linking Surface Receptors Triggered

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (slg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of slg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when slg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was ~0.15 mdyn/pm; for cells treated with antibodies against slg or with Con A the stiffness increased to ~0.6 or 0.4 mdyn/pm, respectively. The stiffness decreased after completion of the capping of slg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against slg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface. Cross-linking lymphocyte surface proteins by multivalent ligands can initiate dramatic changes in the distribution and mobility of cell surface proteins. For example, cross-linking surface immunoglobulin (sIg) ~, the B lymphocyte antigen Abbreviations used in this paper. Anti-IgM, antibody specific for mouse immunoglobulin M; Con A, Concanavalin A; DME-HEPES, Dulbecco's modified Eagle's medium with 20 mM HEPES and 0.1% bovine serum albumin; FITC-Con A, Concanavalin A labeled with fluorescein isothiocyanate; IgM, immunoglobulin M; PBS, phosphate-buffered saline; s-Con A, succinylated Concanavalin A; sIg, lymphocyte surface immunoglobulin; 2xPBS, twofold concentrated phosphate-buffered saline. receptor, by bivalent anti-slg antibodies first immobilizes (1) and then causes an active redistribution of the aggregated ("patched") SIg to one pole of the cell to form a "cap." Subsequently the cell shape changes by formation of a uropod at the site of the cap, and cell motility and chemotaxis can begin (2-4). Evidence from a variety of sources suggests that cytoskeletal contractility is involved in these events. They require cellular energy and can be inhibited by cytochalasins, which interfere with the assembly of actin microfilaments (2). Moreover, a morphological response similar to that which is observed after capping can be simulated by adding ATP in the presence of calcium and magnesium ions to glycerinated THE JOURNAL OF CELL BIOLOGY VOLUME 100 MARCH 1985 860-872 860 © The Rockefeller University Press • 0021-9525185/0310860113 $1.00 on A uust 7, 2017 jcb.rress.org D ow nladed fom lymphocytes (5). Immunofluorescence studies have demonstrated co-localization with both patches and caps of many cytoskeletal components which might be involved in cellular contractility including actin (6, 7), myosin (8, 9), alpha-actinin (10, l l), calmodulin (12, 13), and spectrin (14, 15). More recently, evidence for the participation of myosin light chain kinase in these processes has suggested similarities between the regulation of capping and contraction of smooth muscle (16-18). Other multivalent ligands such as the plant lectin Concanavalin A (Con A), which can bind to a wide range of cell surface glycoproteins, can elicit different but apparently related responses. On cells exposed to Con A at sufficiently high concentration the lectin does not cap, and it inhibits the capping of sIg-anti-sIg aggregates in a process called "anchorage modulation" (19, 20). Moreover, exposure to Con A paralyzes cell motility (21). Inhibition of capping by Con A probably results from retardation of the lateral mobility of the slg molecules (22). Although Con A might bind directly to slg, there is good evidence that its inhibitory effect on the mobility of slg is exerted indirectly through the cytoskeleton. The inhibitory effect is reversed by colchicine and cytochalasin B (22, 23). Furthermore, experiments in which the binding of Con A was restricted to local regions on the lymphocyte surface demonstrate that patching and slg mobility can be retarded at locations on the cell remote from the sites at which the Con A is bound (22, 24, 25). (Different responses to Con A can be observed under other conditions. On cells exposed to low concentrations of Con A [<5 #m/ml] or preincubated at low temperatures or with colchicine [which interferes with microtubule assembly] and then exposed to Con A, Con Aacceptor complexes can cap.) In these responses there seems to be a reflexive pattern in the interactions from plasma membrane to cytoskeleton and then from cytoskeleton back to the plasma membrane. The changes in the cytoskeleton and in surface-cytoskeleton interactions which result from the initial cross-linking can, in turn, influence the mobility and distribution of both the crosslinked and other membrane proteins. Although evidence for the involvement of cytoskeletal contractility in these processes comes from a variety of sources, the direction of cause and effect is not thoroughly established. Conflicting interpretations of the role of the cytoskeleton have been proposed (26, 27). In this study we questioned whether the proposed contractile response of the lymphocyte cytoskeleton to cross-linking surface proteins could be detected by direct mechanical measurements. We expected that the development of substantial contractile forces in the lymphocyte cytoskeletal cortex might exert a circumferential tension around the nucleus which would increase the resistance of the cell to deformation. To test this hypothesis, we have compared the resistance to compression of untreated cells with that of cells on which surface proteins have been cross-linked under conditions which lead to capping or anchorage modulation. This has been accomplished using a recently developed method for measuring the viscoelastic resistance of cells to deformation (28). We have found that there was indeed a substantial cytoskeleton-dependent increase in the resistance of lymphocytes to deformation in cells exposed to surface cross-linking ligands. This elevated resistance decreased after capping or exposure to cytochalasin D or colchicine. Certain ligandindependent treatments, such as elevation of the intracellular calcium ion concentration or exposure to azide ion or hypertonic medium, also increased lymphocyte stiffness. These results demonstrate the use of direct measurements of cellular viscoelasticity as a probe of cellular and cytoskeletal function. MATERIALS AND METHODS