New physical concepts for cell amoeboid motion.

Amoeboid motion of cells is an essential mechanism in the function of many biological organisms (e.g., the regiment of scavenger cells in the immune defense system of animals). This process involves rapid chemical polymerization (with numerous protein constituents) to create a musclelike contractile network that advances the cell over the surface. Significant progress has been made in the biology and biochemistry of motile cells, but the physical dynamics of cell spreading and contraction are not well understood. The reason is that general approaches are formulated from complex mass, momentum, and chemical reaction equations for multiphase-multicomponent flow with the nontrivial difficulty of moving boundaries. However, there are strong clues to the dynamics that allow bold steps to be taken in simplifying the physics of motion. First, amoeboid cells often exhibit exceptional kinematics, i.e., steady advance and retraction of local fixed-shape patterns. Second, recent evidence has shown that cell projections "grow" by polymerization along the advancing boundary of the cell. Together, these characteristics represent a local growth process pinned to the interfacial contour of a contractile network. As such, the moving boundary becomes tractable, but subtle features of the motion lead to specific requirements for the chemical nature of the boundary polymerization process. To demonstrate these features, simple examples for limiting conditions of substrate interaction (i.e., "strong" and "weak" adhesion) are compared with data from experimental studies of yeast particle engulfment by blood granulocytes and actin network dynamics in fishscale keratocytes.     

Fibroblast contractile force is independent of the stiffness which resists the contraction.

Using a device named the cell force monitor, the contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-glycosaminoglycan (GAG) matrices over the first 24 h following cell attachment. In this paper, the effect of a variation in the stiffness that resists matrix contraction by cells on the contractile force generated by the cells was determined. Data from these experiments revealed that the contractile force generated by the fibroblasts was independent of the stiffness of the resistance within the range tested (0.7-10.7 N/m). These results suggest that during the time when fibroblasts are attaching to and spreading on collagen-GAG matrices the contractile forces they generate are force limited, not displacement limited. Therefore, the cytoskeletal mechanism of force generation, corresponding with cell elongation, is capable of increasing the displacement of adhesion sites in order to develop the same level of force. Although a detailed understanding of how the passive mechanical signals provided by substrate materials affect cell processes is still unavailable, in vitro modeling of cell-mediated contraction continues to provide useful information.     

Kinetics of granulocyte phagocytosis: rate limited by cytoplasmic viscosity and constrained by cell size

Micromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37 degrees C; calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37 degrees C to above several min/particle below 15 degrees C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10(-8) cm2 at 37 degrees C to greater than 8 sec/10(-8) cm2 at 15 degrees C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the "apparent" viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-2-glycoprotein 1-dependent macrophage uptake of apoptotic cells. Binding to lipoprotein receptor-related protein receptor family members

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein beta-2-glycoprotein 1 (beta2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of beta2GP1-phosphatidylserine and beta2GP1-apoptotic cell complexes. Using recombinant beta2GP1, we also show that beta2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.     

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative^{1, 2, 3, 4, 5} but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells^{1, 6, 7, 8}; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells⁷; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages ^{9, 10}.In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.     

The role of phagocytes in the protective mechanisms of fish

Phagocytes are cells principally dedicated to the recognition and elimination of invading organisms and damaged tissue. Those described in fish are the granulocytes (particularly neutrophils) and mononuclear phagocytes (tissue macrophages and circulating monocytes). Their movement to sites of microbial invasion is an early event in the inflammatory response and the role of host-derived factors as attractants, such as eicosanoids, is discussed. Opsonins mediate the recognition between phagocyte and particle, and receptors for serum complement component C3 and the Fc fragment of opsonic antibody have been described. Fundamental to the protection offered by the phagocytes is their bactericidal larvacidal activity, which is closely associated with the production of oxygen free radicals. Phagocytes as accessory cells are discussed, including their role in antigen presentation. A knowledge of the modulation of phagocyte function, with activation by various substances and suppression by others, is important if protective responses are to be achieved by up-regulating phagocyte activity.     

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.KEY WORDS: Brain, Intravital microscopy, Leukocytes, Microglia, Neurodegeneration, Zebrafish     

Apoptotic cells induce a phosphatidylserine-dependent homeostatic response from phagocytes

Engulfment of apoptotic cells by phagocytes is important throughout development and adult life. When phagocytes engulf apoptotic cells, they increase their cellular contents including cholesterol and phospholipids, but how the phagocytes respond to this increased load is poorly understood. Here, we identify one type of a phagocyte response, wherein the recognition of apoptotic cells triggers enhanced cholesterol efflux (to apolipoprotein A-I) from macrophages. Phosphatidylserine (PS) exposed on apoptotic cells was necessary and sufficient to stimulate the efflux response. A major mechanism for this enhanced efflux by macrophages was the upregulation of the mRNA and protein for ABCA1, a membrane transporter independently linked to cholesterol efflux as well as engulfment of apoptotic cells. This increase in phagocyte ABCA1 levels required the function of nuclear receptor LXRalpha/beta, a known regulator of cholesterol homeostasis in humans and mice. Taken together, these data reveal a "homeostatic program" initiated in phagocytes that include a proximal membrane signaling event initiated by PS recognition, a downstream signaling event acting through nuclear receptors, and an effector arm involving upregulation of ABCA1, in turn promoting reverse cholesterol transport from the phagocytes. These data also have implications for macrophage handling of contents derived from apoptotic versus necrotic cells in atherosclerotic lesions.     

Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study

We have used an isometric force transducer to study contraction of two types of nonmuscle cells in tissue culture. This method permits the quantitative measurement of contractile force generated by cells of defined type under the influence of external agents while allowing detailed morphological observation. Chick embryo fibroblasts (CEF), which form a contractile network inside a collagen matrix, and human umbilical vein endothelial cells (HUVE), which are located in a monolayer on the surface of the collagen matrix, were studied. CEF and HUVE in 10% FCS produce a substantial tension of 4.5 +/- 0.2 x 10(4) dynes/cm2 and 6.1 x 10(4) dynes/cm2, respectively. Both cell types contract when stimulated with thrombin, generating a force per cell cross-sectional area of approximately 10(5) dynes/cm2, a value approximately an order of magnitude less than smooth muscle. The integrity of the actin cytoskeleton is essential for force generation, as disruption of actin microfilaments with cytochalasin D results in a rapid disappearance of force. Intact microtubules appear to reduce isometric force exerted by CEF, as microtubule-disrupting drugs result in increased tension. Contraction by HUVE precedes a dramatic rearrangement of actin microfilaments from a circumferential ring to stress fibers.     

Molecular and functional characterization of Hv1 proton channel in human granulocytes

Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.     

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation.     

Contact dynamics during keratocyte motility

BACKGROUND: Keratocytes are specialised, rapidly moving cells that generate substantial contractile force perpendicular to their direction of locomotion. Potential roles for contractile force in cell motility include cell-body transport, regulation of adhesion, and retraction of the cell's trailing edge. RESULTS: To investigate contact dynamics, we used simultaneous confocal fluorescence and interference reflection microscopy to image keratocytes injected with fluorescent vinculin. We found that contacts formed behind the leading edge and grew beneath both the lamellipodium and the cell body. Contacts in the middle of the cell remained stationary relative to the substrate and began to disassemble as the cell body passed over them. In contrast, contacts in the lobes of the cell grew continuously and more rapidly, incorporated more vinculin, and slid inwards towards the sides of the cell body. Contact sliding often led to merging of contacts before their removal from the substrate. CONCLUSIONS: We suggest a synthesis of two existing, apparently conflicting models for keratocyte motility, in which network contraction progressively reorients actin filaments using the contacts as pivots, forming bundles that then generate lateral tension by a sliding-filament mechanism. Contact dynamics vary between the middle of the cell and the lobes. We propose that laterally opposed contractile forces first enhance contact growth and stability, but escalating force eventually pulls contacts from the substrate at the back of the cell, without interfering with the cell's forward progress.     

Micromechanics of fibroblast contraction of a collagen-GAG matrix

The contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-GAG matrices over time. We have identified the microscopic deformations developed by individual fibroblasts which lead to the observed macroscopic matrix contraction. Observation of live cells attached to the matrix revealed that matrix deformation occurred as a result of cell elongation. The time dependence of the increase in average fibroblast aspect ratio over time corresponded with macroscopic matrix contraction, further linking cell elongation and matrix contraction. The time dependence of average fibroblast aspect ratio and macroscopic matrix contraction was found to be the result of the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology (2-4 h). The proposed micromechanics associated with observed buckling or bending of individual struts of the matrix by cells may, in part, explain the observation of a force plateau during macroscopic contraction. These findings indicate that the macroscopic matrix contraction measured immediately following cell attachment is related to the extracellular force necessary to support cell elongation, and that macroscopic time dependence is not directly related to microscopic deformation events.     

Unsteady Motion, Finite Reynolds Numbers, and Wall Effect on Vorticella convallaria Contribute Contraction Force Greater than the Stokes Drag

Contraction of Vorticella convallaria, a sessile ciliated protozoan, is completed within a few milliseconds and results in a retraction of its cell body toward the substratum by coiling its stalk. Previous studies have modeled the cell body as a sphere and assumed a drag force that satisfies Stokes' law. However, the contraction-induced flow of the medium is transient and bounded by the substrate, and the maximum Reynolds number is larger than unity. Thus, calculations of contractile force from the drag force are incomplete. In this study, we analyzed fluid flow during contraction by the particle tracking velocimetry and computational fluid dynamics simulations to estimate the contractile force. Particle paths show that the induced flow is limited by the substrate. Simulation-based force estimates suggest that the combined effect of the flow unsteadiness, the finite Reynolds number, and the substrate comprises 35% of the total force. The work done in the early stage of contraction and the maximum power output are similar regardless of the medium viscosity. These results suggest that, during the initial development of force, V. convallaria uses a common mechanism for performing mechanical work irrespective of viscous loading conditions.     

Curvature recognition and force generation in phagocytosis

Background  

The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape.     

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.     

High-speed video cinematographic demonstration of stalk and zooid contraction of Vorticella convallaria.

Stalk contraction and zooid contraction of living Vorticella convallaria were studied by high-speed video cinematography. Contraction was monitored at a speed of 9000 frames per second to study the contractile process in detail. Complete stalk contraction required approximately 9 ms. The maximal contraction velocity, 8.8 cm/s, was observed 2 ms after the start of contraction. We found that a twist appeared in the zooid during contraction. As this twist unwound, the zooid began to rotate like a right-handed screw. The subsequent stalk contraction steps, the behavior of which was similar to that of a damped harmonic oscillator, were analyzed by means of the equation of motion. From the beginning of stalk contraction, the Hookean force constant increased, and reached an upper limit of 2.23 x 10(-4) N/m 2-3 ms after the start of contraction. Thus, within 2 ms, the contraction signal spread to the entire stalk, allowing the stalk to generate the full force of contraction. The tension of an extended stalk was estimated to be 5.58 x 10(-8) N from the Hookean force constant of a stalk. This value coincides with that of the isometric tension of a glycerol-treated V. convallaria, confirming that the contractile system of V. convallaria is well preserved despite glycerol treatment.     

BAD1, an essential virulence factor of Blastomyces dermatitidis,suppresses host TNF-alpha production through TGF-beta-dependent and -independent mechanisms

We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-alpha production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-beta in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-beta mAb neutralized detectable TGF-beta and restored phagocyte TNF-alpha production. Similarly, addition of anti-TGF-beta mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-alpha production. Conversely, TGF-beta treatment of phagocytes cultured with knockout yeast suppressed TNF-alpha production. Hence, TGF-beta mediates BAD1 suppression of TNF-alpha by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-beta levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-alpha levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-alpha production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-beta, soluble BAD1 failed to do so and TNF-alpha suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-beta. Thus, BAD1 of B. dermatitidis induces suppression of TNF-alpha and progressive infection by both TGF-beta-dependent and -independent mechanisms.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

The regional association of actin and myosin with sites of particle phagocytosis

Contractile proteins are thought to play a causative role in motile processes such as phagocytosis. In order to investigate their role in phagocytosis further, simultaneous immunofluorescence localization of F-actin and myosin was carried out in resident mouse peritoneal macrophages after phagocytosis of opsonized zymosan particles. Both actin and myosin appeared to concentrate rapidly at sites of particle phagocytosis. The observed concentration of both proteins at such sites preceded ultimate particle engulfment. Cytochalasin B, a drug which was shown to block pseudopod extensions around the particle, did not prevent the concentration of the two contractile proteins at cell-particle binding sites. This result ruled out path-length effects as an explanation for the observed concentration of actin and myosin at phagocytic sites. Kinetic analysis showed that actin rapidly concentrates at particle-cell binding sites within minutes (or less) of contact with cell surface. The two proteins are present throughout the engulfment phase until and after ingestion is complete. Finally, at later times the particles become clustered over the cell nucleus and the particle-associated actin-myosin seen earlier is no longer evident.