Cytosolic free Ca2+ changes and calpain activation are required for β integrin–accelerated phagocytosis by human neutrophils

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca2+ imaging, the temporal relationship between changes in cytosolic free Ca2+ concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca2+ changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca2+ which resulted from Ca2+ influx. This latter rise in cytosolic free Ca2+ concentration also liberated a fraction of β2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized β2 integrin labelling. Inhibitors of calpain activation prevented both the Ca2+-induced liberation of β2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca2+ signal. Therefore, we propose that Ca2+ activation of calpain causes β2 integrin liberation, and that this signal plays a key role in the acceleration of β2 integrin–mediated phagocytosis

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

Minimal impact electro-injection of cells undergoing dynamic shape change reveals calpain activation

AbstractThe ability of neutrophils to rapidly change shape underlies their physiological functions of phagocytosis and spreading. A major problem in establishing the mechanism is that conventional microinjection of substances and indicators interferes with this dynamic cell behaviour. Here we show that electroinjection, a “no-touch” point-and-shoot means of introducing material into the cell, is sufficiently gentle to allow neutrophils to be injected whilst undergoing chemokinesis and spreading without disturbing cell shape change behaviour. Using this approach, a fluorogenic calpain-1 selective peptide substrate was introduced into the cytosol of individual neutrophils undergoing shape changes. These data showed that (i) physiologically elevated cytosolic Ca2+ concentrations were sufficient to trigger calpain-1 activation, blockade of Ca2+ influx preventing calpain activation and (ii) calpain-1 activity was elevated in spreading neutrophil. These findings provide the first direct demonstration of a physiological role for Ca2+ elevation in calpain-1 activation and rapid cell spreading. Electroinjection of cells undergoing dynamic shape changes thus opens new avenues of investigation for defining the molecular mechanism underlying dynamic cell shape changes

Translocation or just location? Pseudopodia affect fluorescent signals

The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe

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

Field Measurements of Terrestrial and Martian Dust Devils

Surface-based measurements of terrestrial and martian dust devils/convective vortices provided from mobile and stationary platforms are discussed. Imaging of terrestrial dust devils has quantified their rotational and vertical wind speeds, translation speeds, dimensions, dust load, and frequency of occurrence. Imaging of martian dust devils has provided translation speeds and constraints on dimensions, but only limited constraints on vertical motion within a vortex. The longer mission durations on Mars afforded by long operating robotic landers and rovers have provided statistical quantification of vortex occurrence (time-of-sol, and recently seasonal) that has until recently not been a primary outcome of more temporally limited terrestrial dust devil measurement campaigns. Terrestrial measurement campaigns have included a more extensive range of measured vortex parameters (pressure, wind, morphology, etc.) than have martian opportunities, with electric field and direct measure of dust abundance not yet obtained on Mars. No martian robotic mission has yet provided contemporaneous high frequency wind and pressure measurements. Comparison of measured terrestrial and martian dust devil characteristics suggests that martian dust devils are larger and possess faster maximum rotational wind speeds, that the absolute magnitude of the pressure deficit within a terrestrial dust devil is an order of magnitude greater than a martian dust devil, and that the time-of-day variation in vortex frequency is similar. Recent terrestrial investigations have demonstrated the presence of diagnostic dust devil signals within seismic and infrasound measurements; an upcoming Mars robotic mission will obtain similar measurement types

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

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