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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. [ABSTRACT FROM AUTHOR]

Studies calcium ion influx shutdown during neutrophil apoptosis. Goals of neutrophil apoptosis; Unregulated and regulated neutrophil apoptosis; Link between mitochondria in apoptosis and control of calcium ion influx.

Cytosolic free Ca2+and intranuclear Ca2+behave similarly in human neutrophils. However, conventional laser scanning at 350 ms/frame resolution at lower than physiological temperatures demonstrates that (i) the nuclear fluo3–Ca2+signal persists longer than the cytosolic signal in some (but not all) nuclear lobes, (ii) the neutrophil nuclear membrane and fine inter-lobe filaments present barriers to diffusion of fluo3–Ca2+and lucifer yellow, and (iii) the diffusion barrier correlates with condensed chromosomal material on the nuclear envelope and blockage of the movement of fluo3–Ca2+into individual nuclear lobes. [Copyright &y& Elsevier]

Examines the mechanism of phagocytosis of microbes coated with opsonin using calpain activation and cystolic free calcium ions. Localization of calcium changes at the site of particle attachment; Need for a rapid engulfment of particles required for global changes in cystolic free calcium ions; Liberation of beta2 integrin receptors.

Abstract: The first report that high speed Ca2+ waves travel in a restricted zone around the cell periphery in a clockwise manner and which can only be revealed by very high speed imaging has been quietly retracted. In the original report, a single small region of high Ca2+ was seen to spin around the cell periphery and around phagosomes formed within phagocytes in a manner unlike anything reported previously. This consequently caused a lot of interest. Several other papers reporting a similar phenomenon in a variety of cells were also published by the same lab and the phenomenon has been included in high profile reviews such as in Science. However, several groups have failed to reproduce this work and attempts to detect the Ca2+ waves or the mechanism for their generation have failed. Now, the original report has been quietly retracted. In this short commentary, we give a brief account of the sorry story of the non-existent circulating Ca2+ waves (z-waves) in the hope that the literature can be rectified and that future cell calcium biologists are not misled by these fantastic and spurious Ca2+ reports. [Copyright &y& Elsevier]

Abstract: The 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. [Copyright &y& Elsevier]