Intracellular vesicle movement, cAMP and myosin II in Dictyostelium

Dictyostelium amoebae were analyzed before and after rapid addition of 10(-6) M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F-actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10(-6) M cAMP addition, cells stopped translocating, pseudopod formation ceased, intracellular particle movement was depressed, and F-actin was lost from the pseudopod and concomitantly relocalized in the cell cortex. After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilamellar membrane bodies. The possible role of these vesicles is discussed.     

Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement

Both cellular motility and intracellular particle movement are compared between normal Dictyostelium amebae of strain AX4 and amebae of a myosin II heavy chain null mutant, HS2215, using the computer assisted "Dynamic Morphology System." In AX4 cells rapidly translocating in buffer, cytoplasmic expansion is apical and the majority of intracellular particles move anteriorly, towards the site of expansion. When these cells are pulsed with 10(-6) M cAMP, the peak concentration of the natural cAMP wave, cells stop translocating and average particle velocity decreases threefold within 2-4 s after cAMP addition. After 8 s, there is a partial rebound both in cytoplasmic expansion and particle velocity, but in both cases, original apical polarity is lost. In HS2215 cells in buffer, both cellular translocation and average particle velocity are already at the depressed levels observed in normal cells immediately after cAMP addition, and no anterior bias is observed in either the direction of cytoplasmic expansion or the direction of particle movement. The addition of cAMP to myosin-minus cells results in no additional effect. The results demonstrate that myosin II is necessary for (a) the rapid rate of intracellular particle movement, (b) the biased anterior directionality of particle movement, and (c) the rapid inhibition of particle movement by cAMP.     

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility.

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB-). DMIB- cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB- cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB- cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB- cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB- cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB- cells.     

Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.     

Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave.

The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer-assisted dynamic image analyzing system. During the first approximately 150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a "freeze" in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase.     

Pseudopodium dynamics and rapid cell movement in Dictyostelium Ras pathway mutants

Loss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility that underlie these phenotypes using computer-assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS(-) cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB(-) cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F-actin in both mutant cell lines are significantly elevated with respect to the wild-type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes.     

A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

Increased intracellular H(+) efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1(-)) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1(-) cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI((3,4,5))P(3)). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI((3,4,5))P(3) production and for attaining a polarized phenotype.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

A Rho GTPase Signal Treadmill Backs a Contractile Array

Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying "signal treadmill" in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2-powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays, and proper functioning of these gradients is dependent on both contraction and F-actin turnover. VIDEO ABSTRACT:     

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.     

Minimal amino acids in the I/LWEQ domain required for anterior/posterior localization in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Establishment of cell polarity is mediated by a series of signaling molecules that are asymmetrically activated or localized in the cell upon extracellular stimulation. To understand the mechanism that mediates anterior/posterior asymmetric localization of RapGAP3 during migration, we determined the minimally required amino acids in the I/LWEQ domain that cause posterior localization and found that the minimal region of the F-actin binding domain for posterior localization could, with some additional deletion at the C-terminal, localize to the anterior. Analysis of the localization and translocation kinetics to the cell cortex of the truncated proteins suggests that the required regions for anterior/posterior localization might have a preferential binding affinity to preexisting F-actins at the rear and lateral sides of the cell or newly formed F-actins at the front of the cell, leading to distinct differential sites of the cell.     

Dual chemotaxis signalling regulates Dictyostelium development: intercellular cyclic AMP pulses and intracellular F-actin disassembly waves induce each other

Aggregating Dictyostelium discoideum amoebae periodically emit and relay cAMP, which regulates their chemotaxis and morphogenesis into a multicellular, differentiated organism. Cyclic AMP also stimulates F-actin assembly and chemotactic pseudopodium extension. We used actin-GFP expression to visualise for the first time intracellular F-actin assembly as a spatio-temporal indicator of cell reactions to cAMP, and thus the kinematics of cell communication, in aggregating streams. Every natural cAMP signal pulse induces an autowave of F-actin disassembly, which propagates from each cell's leading end to its trailing end at a linear rate, much slower than the calculated and measured velocities of cAMP diffusion in aggregating Dictyostelium. A sequence of transient reactions follows behind the wave, including anterior F-actin assembly, chemotactic pseudopodium extension and cell advance at the cell front and, at the back, F-actin assembly, extension of a small retrograde pseudopodium (forcing a brief cell retreat) and chemotactic stimulation of the following cell, yielding a 20s cAMP relay delay. These dynamics indicate that stream cell behaviour is mediated by a dual signalling system: a short-range cAMP pulse directed from one cell tail to an immediately following cell front and a slower, long-range wave of intracellular F-actin disassembly, each inducing the other.     

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

A three-component mechanism for fibroblast migration with a contractile cell body that couples a myosin II-independent propulsive anterior to a myosin II-dependent resistive tail

To understand the mechanism of cell migration, we cultured fibroblasts on micropatterned tracks to induce persistent migration with a highly elongated morphology and well-defined polarity, which allows microfluidic pharmacological manipulations of regional functions. The function of myosin II was probed by applying inhibitors either globally or locally. Of interest, although global inhibition of myosin II inhibited tail retraction and caused dramatic elongation of the posterior region, localized inhibition of the cell body inhibited nuclear translocation and caused elongation of the anterior region. In addition, local application of cytochalasin D at the tip inhibited frontal extension without inhibiting forward movement of the cell nucleus, whereas local treatment posterior to the nucleus caused reversal of nuclear movement. Imaging of cortical dynamics indicated that the region around the nucleus is a distinct compression zone where activities of anterior and posterior regions converge. These observations suggest a three-component model of cell migration in which a contractile middle section is responsible for the movement of a bulky cell body and the detachment/retraction of a resistive tail, thereby allowing these regions to undergo coordinated movement with a moving anterior region that carries little load.     

The role of myosin II motor activity in distributing myosin asymmetrically and coupling protrusive activity to cell translocation

Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at > or = 30 microM inhibited anterior redistribution of myosin IIA, with 100 microM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.     

Myosin II dynamics in Dictyostelium: determinants for filament assembly and translocation to the cell cortex during chemoattractant responses

In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G-protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP-myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP-myosin fusions, and with latrunculin A-treated cells, demonstrated that F-actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A "headless" GFP-myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor-based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant-stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant-regulated myosin II filament assembly and translocation.     

Targeted disruption of the ABP-120 gene leads to cells with altered motility

The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).     

Proteasomal activity modulates TGF-ss signaling in a gene-specific manner

Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.     

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, “classical chemoattractants” such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and “pure chemoattractants” such as TGF-β1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the “non-classical” group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca²⁺]_i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca²⁺-independent G-protein-coupled pathway for post-receptor signal transduction triggered by “pure chemoattractants.” The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds. © 1996 Wiley-Liss, Inc.