Intraphagosomal Free Ca2+ Changes during Phagocytosis.

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.

Localisation of Intracellular Signals and Responses during Phagocytosis.

Phagocytosis is one of the most polarised of all cellular activities. Both the stimulus (the target for phagocytosis) and the response (its internalisation) are focussed at just one part of the cell. At the locus, and this locus alone, pseudopodia form a phagocytic cup around the particle, the cytoskeleton is rearranged, the plasma membrane is reorganised, and a new internal organelle, the phagosome, is formed. The effect of signals from the stimulus must, thus, both be complex and yet be restricted in space and time to enable an effective focussed response. While many aspects of phagocytosis are being uncovered, the mechanism for the restriction of signalling or the effects of signalling remains obscure. In this review, the details of the problem of restricting chemical intracellular signalling are presented, with a focus on diffusion into the cytosol and of signalling lipids along the plasma membrane. The possible ways in which simple diffusion is overcome so that the restriction of signalling and effective phagocytosis can be achieved are discussed in the light of recent advances in imaging, biophysics, and cell biochemistry which together are providing new insights into this area.

EPIC3, a novel Ca2+ indicator located at the cell cortex and in microridges, detects high Ca2+ subdomains during Ca2+ influx and phagocytosis.

The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally-induced Ca2+influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30-50 µM, with peak locations towards the tips of wrinkles reaching 80 µM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50-80 µM) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils.

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca2+ signal in osmotically shrunk cells (by IP3 uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca2+ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.

An Introduction to Phagocytosis.

Phagocytosis is usually defined as the cellular process by which cells internalise particulate matter larger than about 0.5 µm in diameter. It is an endocytic process, distinct from pinocytosis and macropinocytosis. These latter processes may internalise small particles suspended the extracellular fluid, but this is a by-product of internalising the fluid, and is not phagocytosis per se. In contrast, phagocytosis is targeted at solid particulates, usually microbes, which are internalised and "digested" either to provide food, or as part of the immune system of higher animals. The mechanism of phagocytosis may have, at its core, many primitive elements, but it is a highly complex and coordinated series of cell biological and molecular events which together result in the uptake of a particle. In this introduction, the basis of phagocytosis and some ideas of its origin are discussed.

A Brief History of Phagocytosis.

This chapter outlines some of the more significant steps in our understanding of the phenomenon and mechanism of phagocytosis. These are mainly historical, ranging from near the advent of microscopy in the seventeenth and eighteenth century up to the period before the Second World War (1930s). During this time, science itself moved from being the domain of the wealthy enthusiast to the professional and funded university scientist. Not surprisingly progress was slow of the first two centuries of phagocytic research, but accelerated around the late nineteenth century and the turn of the twentieth century. Since then progress has accelerated still further. This chapter however aims to put our current progress into a historical context and to explore some of the interesting personalities who have set the ground work for our current understanding of the subject of this book, namely phagocytosis.

Membrane Tension and the Role of Ezrin During Phagocytosis.

During phagocytosis, there is an apparent expansion of the plasma membrane to accommodate the target within a phagosome. This is accompanied (or driven by) a change in membrane tension. It is proposed that the wrinkled topography of the phagocyte surface, by un-wrinkling, provides the additional available membrane and that this explains the changes in membrane tension. There is no agreement as to the mechanism by which unfolding of cell surface wrinkles occurs during phagocytosis, but there is a good case building for the involvement of the actin-plasma membrane crosslinking protein ezrin. Not only have direct measurements of membrane tension strongly implicated ezrin as the key component in establishing membrane tension, but the cortical location of ezrin changes at the phagocytic cup, suggesting that it is locally signalled. This chapter therefore attempts to synthesise our current state of knowledge about ezrin and membrane tension with phagocytosis to provide a coherent hypothesis.

Calpain Activation by Ca2+ and Its Role in Phagocytosis.

Although the cytosolic Ca2+ signalling event in phagocytosis is well established, and the mechanism for generating such signals also understood, the target for the Ca2+ signal and how this relates to the phagocytic outcome is less clear. In this chapter, we present the evidence for a role of the Ca2+ activated protease, calpain, in phagocytosis. The abundant evidence for Ca2+ changes and calpain activation during cell shape changes is extended to include the specific cell shape change which accompanies phagocytosis. The discussion therefore includes a brief description of the domain structure of calpain and their functions. Also the mechanism by which calpain activation is limited at the cell periphery subdomains, and how this would allow phagocytic pseudopodia to form locally.

Conclusions and the Futures of Phagocytosis.

Although we know a wealth of detail about the molecular and cell biology of phagocytosis, there are many unsolved mysteries remaining. In this final chapter, some important may be tangential) questions are raised, that the bulk of researchers are not really addressing. In this chapter, some suggestions are given for this type of "blue skies" future work. These include new approaches to understanding phagocytosis and the possibility that this new knowledge may provide a solution to anti-microbial resistance. This future phagocytosis research would have an impact, not only on our understanding of phagocytosis, but potentially on the future of human health.

Ca2+-activated cleavage of ezrin visualised dynamically in living myeloid cells during cell surface area expansion.

The intracellular events underlying phagocytosis, a crucial event for innate immunity, are still unresolved. In order to test whether the reservoir of membrane required for the formation of the phagocytic pseudopodia is maintained by cortical ezrin, and that its cleavage is a key step in releasing this membrane, the cleavage of cortical ezrin was monitored within living phagocytes (the phagocytically competent cell line RAW264.7) through expressing two ezrin constructs with fluorescent protein tags located either inside the FERM or at the actin-binding domains. When ezrin is cleaved in the linker region by the Ca2+-activated protease calpain, separation of the two fluorophores would result. Experimentally induced Ca2+ influx triggered cleavage of peripherally located ezrin, which was temporally associated with cell expansion. Ezrin cleavage was also observed in the phagocytic pseudopodia during phagocytosis. Thus, our data demonstrates that peripheral ezrin is cleaved during Ca2+-influx-induced membrane expansion and locally within the extending pseudopodia during phagocytosis. This is consistent with a role for intact ezrin in maintaining folded membrane on the cell surface, which then becomes available for cell spreading and phagocytosis.

Microinjection and Micropipette-Controlled Phagocytosis Methods for Neutrophils.

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization.

Optical Methods for the Measurement and Manipulation of Cytosolic Calcium Signals in Neutrophils.

The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils, and are discussed here, especially the use of fluorescent indicators of Ca2+ and photoactivation of reagents involved in Ca2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we outline some of the techniques that have been used to monitor, visualize, and manipulate Ca2+ in neutrophils.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events.

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

Neutrophil Cell Shape Change: Mechanism and Signalling during Cell Spreading and Phagocytosis.

Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role-killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.

Defective rapid cell shape and transendothelial migration by calpain-1 null neutrophils.

It has been proposed that Ca2+ activation of calpain-1 is an important trigger for rapid cell spreading by neutrophils. In this paper, we have investigated this by assessing the ex vivo functioning of neutrophils from calpain-1 null mice, Calpain-1 null neutrophils failed to migrate through TNF-activated endothelial monolayers. The failure to transmigrate through endothelial monolayers was therefore unlikely to be due to a failure of chemotaxis as chemotaxis by adherent calpain-1 null neutrophils towards fMLP was unpaired. In contrast, the capacity of calpian-1 neutrophils to spontaneously spread was limited to smaller diameters than for wild type cells. Photolytic uncaging of IP3 with Individual wild type neutrophils resulted in a large Ca2+ signal and rapid cell spreading. In contrast, calpain-1 neutrophils failed to spread in response to the IP3-induced Ca2+ signal. This work has therefore demonstrated that the presence of calpain-1 was required for effective rapid cell spreading by neutrophils.

Phagocytosis and Motility in Human Neutrophils is Competent but Compromised by Pharmacological Inhibition of Ezrin Phosphorylation.

BACKGROUND AND OBJECTIVE: Ezrin links the cortical cytoskeleton to the plasma membrane and plays a role in regulating changes in cell shape. Recently, NSC668394 has been shown to inhibit a key step for its activity, i.e. phosphorylation at threonine 567. In neutrophils, another key regulatory step is the Ca2+-mediated cleavage of ezrin by calpain. METHODS: In this paper, we use NSC668394 as a pharmacological inhibitor to investigate the interplay between these two steps in regulating changes in neutrophil shape. RESULTS: NSC668394 reduced the amount of peripherally located ezrin in neutrophils, and increased Ca2+-dependent ezrin cleavage. Neutrophils with NSC668394-inhibited ezrin phosphorylation remained both phagocytic and chemotactically competent. However, phagocytosis was slightly impaired and chemotaxis could not be maintained over longer periods. The characteristic chemotactic morphology which neutrophils adopt was also aberrant. Although phosphorylation of ezrin plays a minor role in limiting the rapid changes in cell shape in neutrophils, inhibition of ezrin phosphorylation by NSC668394 prevented multiple and prolonged shape changes during extended chemotaxis. CONCLUSION: The susceptibility of prolonged chemotaxis to inhibition by NSC668394 may point to a useful target for anti-inflammatory therapy. Inhibition of neutrophil chemotaxis towards chronically inflamed sites without compromising their ability to undergo phagocytosis is a much sought after the effect of anti-neutrophil therapy.

Topographical interrogation of the living cell surface reveals its role in rapid cell shape changes during phagocytosis and spreading.

Dramatic and rapid changes in cell shape are perhaps best exemplified by phagocytes, such as neutrophils. These cells complete the processes of spreading onto surfaces, and phagocytosis within 100 s of stimulation. Although these cell shape changes are accompanied by an apparent large increase in cell surface area, the nature of the membrane "reservoir" for the additional area is unclear. One proposal is that the wrinkled cell surface topography (which forms micro-ridges on the neutrophil surface) provides the resource for neutrophils to expand their available surface area. However, it has been problematic to test this proposal in living cells because these surface structures are sub-light microscopic. In this paper, we report the development of a novel approach, a variant of FRAP (fluorescent recovery after photo-bleaching) modified to interrogate the diffusion path-lengths of membrane associated molecules. This approach provides clear evidence that the cell surface topography changes dramatically during neutrophil shape change (both locally and globally) and can be triggered by elevating cytosolic Ca2+.

Active calpain in phagocytically competent human neutrophils: electroinjection of fluorogenic calpain substrate.

Calpain has been implicated in the apparent expansion of neutrophil plasma membrane that accompanies cell spreading and phagocytosis. In order to test this hypothesis, an internally quenched fluorescent peptide substrate of calpain-1 which increased in fluorescence on cleavage, was micro-electroinjected into neutrophils. The fluorescence intensity increased in a significant number of neutrophils, including those which appeared to be in a morphologically resting (spherical) state. In order to test whether calpain was activated by an elevation of cytosolic Ca(2+) during the injection, Ca(2+) chelators were added to the injectate and cytosolic free Ca(2+) in the receiving neutrophil was simultaneously monitored. It was shown that this approach could be used without raising Ca(2+) within the injected cell. Despite this, approximately 75% of individual neutrophils had calpain activity which consumed the substrate within approx. 100 s. It was found that all neutrophils had elevated calpain activity were phagocytically competent; whereas neutrophils with low or undetectable calpain activity failed to undergo phagocytosis. This association was consistent with the hypothesis that calpain activity within neutrophils was necessary for them to undergo efficient phagocytosis.

The structural basis of differential inhibition of human calpain by indole and phenyl α-mercaptoacrylic acids.

Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer's disease. Calpain-I is a Ca(2+)-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these compounds represent promising drug leads. Here we present the 2.05 and 2.03 Å co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid. In both structures, the α-mercaptoacrylic acid derivatives bind between two α-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring.