Tim Whalley - Academia.edu (original) (raw)

Papers by Tim Whalley

Cold Spring Harbor Symposia on Quantitative Biology, 1995

Biochimica et biophysica acta. Molecular cell research, Sep 1, 1991

In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulator... more In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulatory actions, although its presence (and by implication, phosphorylation) is not obligatory for s zretion to occur. These effects include (!) the enhancement of the sensi;P~|ty to both of the essential effectors (Ca 2+ and guanine nucleotide); (2) the maintenance of the responsiveness of permeabilised cells; (3) restoration of responsiveness to cells rendered refractory by previous permeabilisatiou, and (4) induction of delays in the onset of exocytosis from permeabilised cells. We define the modulatory reactions induced by ATP by characterising their specificity to other potential phosphorylating nucleotides and their requirement for Mg z+. GTP and AppNHp are without effect in any of the modulatory actions. ATP, ATP-y-S, ITP, XTP, CTP and UTP all appear to support an enhancement of the sensitivity to GTP-y-S when applied immediately at the time of permeabilisation. Hog, vet, the non.adenine nucleoside triphosphates appear to mediate their effect by transphosphorylation to ADP, and therefore the active species appears to be ATP. Only ATP is capable of maintaining and restoring responsiveness (2 and 3 above). Only ATP and ATP-y-S induce onset delays and do so moreover in the absence (< 10-s M) of Mg a+. We conclude that three of the modulatory effects (1, 2 and 3 above) which all express a requirement l'or Mg 2+, and can be prevented by inhibitors of protein kinase C are likely to result from phosphorylation reactions. The induction of delays by ATP

Molecular Biology of the Cell, Mar 1, 1992

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by... more We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca21]i). The increase in [Ca21]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca21]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,NN'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca21]i increase and activation are prevented in eggs in which the InsP3sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

International Archives of Allergy and Immunology, 1991

The release of inflammatory mediators from mast cells occurs by a regulated exocytotic process. W... more The release of inflammatory mediators from mast cells occurs by a regulated exocytotic process. We have been able to study the intracellular events in this pathway by permeabilising the plasma membrane of rat peritoneal mast cells and stimulating exocytosis by providing both Ca2+ and a guanine nucleotide. By this approach we have obtained evidence for the participation of at least two guanine nucleotide binding proteins in the control of exocytosis. We have also shown that ATP is unnecessary for the final events, but that it does have a number of modulatory functions, for instance in the control of the effective affinity of the proteins that bind Ca2+ and GTP. There is also evidence for a protein dephosphorylation in the later stages of the control pathway.

Bioscience Reports, Aug 1, 1988

Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by reco... more Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to i raM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mMCa before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back fusion of secretory granules that are in contact with the plasma membrane. Our results are also consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic protein.

Proceedings of the National Academy of Sciences of the United States of America, Jun 21, 1994

Exit from mitosis requires inactivation of the cycin B-p34akd protein kinase complex. Since e d c... more Exit from mitosis requires inactivation of the cycin B-p34akd protein kinase complex. Since e d cytosolic Ca2+ has been niplicated as a potential trigger of nitotic ogression, we directly tested the possib~ty that Ca+ triggers the pathway resonibl for inactivatin the cdc2 kina, using sea urchin embryos pe ed at various stages of the cell cycle. In cells permeabilized during late interphase and prophase, micromolar Ca2+ induced premature inactivation of the cdc2 kinase without fig th absolute amount ofp34aw protein. Inactivation was selective for the cdc2 kinase, as elevated Ca+ had no effect on cAMP-dependent protein kinase activity. Premature cdc2 kinase inactivation did not require cydlin B destruction, but did coincide with the d atio of cycl p34°d c plexes. In cells perneabilize during and metaphase, cdc2 kinase inactivation was Ca2+-deprndea~t, presumably because at these later times the inactivaing pathway had been enabled prior to permealtion. This work provides evidence that Ca+ is the physiological igger enablng cdc2 kinase inactivation during mitosis.

Journal of Invertebrate Pathology, Nov 1, 2012

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlant... more Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.

Journal of Biological Chemistry, Dec 1, 1998

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate t... more The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (Ϸ70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca 2؉ ] free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca 2؉-triggering step, blocks complex disruption by Ca 2؉. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca 2؉-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca 2؉ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca 2؉ binding removes a "fusion-clamp." Membrane fusion is the fundamental cellular process by which exocytotic secretion, enveloped virus entry, intracellular trafficking, and fertilization occur. Recently, conceptual advances have been made in how we think of the proteins involved in intracellular membrane trafficking and exocytosis (1-3), due in part to the discovery of a series of interactions between homologous proteins known to be required for membrane trafficking in vitro, yeast secretion in vivo, and synaptic transmission at the neuromuscular junction. These interactions are thought to contribute to the formation of a protein complex that is postulated to mediate the targeting, docking, and subsequent fusion of membranes (2).

Journal of Cell Biology, Dec 1, 1995

At fertilization in sea urchin eggs, elevated cytosolic Ca 2+ leads to the exocytosis of 15,000-1... more At fertilization in sea urchin eggs, elevated cytosolic Ca 2+ leads to the exocytosis of 15,000-18,000 1.3-1xm-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueousand lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-1xm-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 rain. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrincoated vesicles. When eggs are treated with short wavelength ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.

Biochemical Journal, Nov 15, 1995

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required fo... more The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent Nethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component * To whom corresp.ondence should be addressed.

American Journal of Physiology-cell Physiology, Jun 1, 2017

2016.-Mutations in the gene that encodes the principal L-carnitine transporter, OCTN2, can lead t... more 2016.-Mutations in the gene that encodes the principal L-carnitine transporter, OCTN2, can lead to a reduced intracellular L-carnitine pool and the disease Primary Carnitine Deficiency. L-Carnitine supplementation is used therapeutically to increase intracellular L-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase L-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase L-carnitine uptake at 100 nM. However, L-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 M)], inhibit mitochondrial function [sodium azide (75 M), rotenone (1 M), berberine (100 M), DNP (500 M)], or directly activate AMPK [AICAR (250 M)] were assessed for their ability to regulate L-carnitine uptake. All compounds tested significantly inhibited L-carnitine uptake. Inhibition by caffeine was not dantrolene (10 M) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit L-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 M) to rescue the effect of caffeine. Compound C offered a partial rescue of L-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits L-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role. carnitine uptake; AMPK; insulin; kinase assays L-CARNITINE [3-hydroxy-4-(trimethylazaniumyl) butanoate] is a dipeptide compound which acts as a cofactor for the transport of long chain fatty acids into the mitochondria where they can * A. Shaw and S. Jeromson contributed equally to this work.

Biochemical Journal, Sep 1, 1994

It is known that sea-urchin egg cortical-granule exocytosis is inhibited by agents such as N-ethy... more It is known that sea-urchin egg cortical-granule exocytosis is inhibited by agents such as N-ethylmaleimide (NEM) which modify thiol groups. The fusion-related proteins modified by these agents have yet to be identified, nor is there information regarding the topography of these thiol groups. Furthermore, the step in cortical-granule exocytosis at which these thiol groups participate is unknown. In this study we have investigated the topological properties of, and the temporal requirement for the function of, the fusion-related thiol groups by treating the isolated exocytotic apparatus with high-molecular-mass dextrans and BSA carrying thiol-reactive 3-(2-pyridyldithio)propionate groups. The dextran derivatives inhibited exocytosis. The BSA

Journal of Cell Science, May 1, 2004

Introduction Cortical vesicles (CVs) are protein-filled secretory vesicles found in most animal e... more Introduction Cortical vesicles (CVs) are protein-filled secretory vesicles found in most animal eggs (Peres and Bernardini, 1985; Chandler, 1991; Tahara et al., 1996). At fertilization, CVs undergo exocytosis in response to the elevated cytosolic free Ca 2+ concentration ([Ca 2+ ] free) triggered by gamete fusion (Swann and Whitaker, 1986). In sea urchin eggs, the fully primed CVs exist, already docked to the plasma membrane (PM), and disruption of unfertilized eggs in Ca 2+-free medium produces sheets of plasma membrane with the CVs firmly attached (Vacquier, 1975; Zimmerberg et al., 1985). In such an egg cortex preparation, exocytosis is triggered by the addition of micromolar [Ca 2+ ]free (Baker and Whitaker, 1978; Sasaki and Epel, 1983; Zimmerberg and Liu, 1988; Blank et al., 1998), and this ionic signal is the only requirement for fusion in vitro (Whitaker, 1987). In addition to fusion with the PM, isolated CVs can also fuse with each other (Vogel and Zimmerberg, 1992) in response to micromolar concentrations of Ca 2+ , showing that the vesicles themselves possess the entire machinery required for Ca 2+-dependent membrane fusion. Characterization of the mechanism of CV-CV fusion has shown that it is the same as for exocytosis in vitro (Coorssen et al., 1998). A central issue that remains controversial concerns the identity of the protein components of the intracellular membrane fusion machine. Several proteins play important roles in the movement of proteins between the endoplasmic reticulum and other intracellular compartments, pathways that also involve membrane fusion (Rothman, 1994; Lin and Scheller, 2000; Brunger, 2001). These include the cytosolic proteins N-ethylmaleimide (NEM)-sensitive fusion protein (NSF), a hexameric ATPase (Whiteheart et al., 1994; Tagaya et al., 1993; Clary and Rothman, 1990) and soluble NSFattachment proteins or SNAPs (Clary and Rothman, 1990; Clary et al., 1990); as well as a complex of membrane-bound proteins called SNAP receptors or SNAREs (Wilson et al., 1992; Whiteheart et al., 1992). A heterotrimeric complex of SNARE proteins is reportedly able to catalyze fusion of liposomes (Weber et al., 1998), although evidence from some physiologically relevant biological systems seems to indicate that SNARE protein complexes are not involved in the final stages of membrane fusion in native membranes (Coorssen et al., 1998; Ungermann et al., 1998a; Ungermann et al., 1998b; Peters et al., 2001). Indeed, recent work that has analyzed the sensitivity of SNARE proteins and Ca 2+-triggered fusion to a variety of proteases or to blockade by exogenous SNARE binding proteins, suggests that membrane fusion requires additional proteins that function 2345 The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca 2+-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-γ-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca 2+-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

Journal of Cell Biology, May 15, 1991

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urch... more We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATE ATPTS (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinase s, leading to irreversible protein thiophosphorylation (Gratecos, D., and E. H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP3,S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATPTS requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATPTS irreversibly inhibits exo

The conversation, Jun 14, 2016

Biochemical Journal, Jul 10, 2000

The p34 cdc# protein kinase, a universal regulator of mitosis, is controlled positively and negat... more The p34 cdc# protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34 cdc# are induced by Ca# + and prevented by Ca# + chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca# + transients may play an important physiological role in the control of p34 cdc# kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca# + concentration ([Ca# + ] i) in early sea urchin embryos without altering the normal resting level of Ca# +. We have used this finding to investigate whether [Ca# + ] i transients control p34 cdc# kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34 cdc#. In the present study we show that the elimination of [Ca# + ] i transients during interphase blocks p34 cdc# activation and entry into mitosis, while the Abbreviations used : BAPTA, bis-(o-aminophenoxy)ethane-N,N,Nh,Nh-tetra-acetic acid ; [Ca 2 + ] i , intracellular Ca 2 + concentration ; diC 8 , 1,2-dioctanoylsn-glycerol ; CFASW, calcium-free artificial sea water ; DTT, dithiothreitol ; NEBD, nuclear envelope breakdown ; PKC, protein kinase C ; PLC, phospholipase C ; TCA, trichloroacetic acid. 1 To whom correspondence should be addressed (e-mail tdw1!stir.ac.uk).

Cell regulation, Feb 1, 1991

We show that microinjecting guanosine-5'-thiotriphosphate (GTPyS) into unfertilized sea urchin eg... more We show that microinjecting guanosine-5'-thiotriphosphate (GTPyS) into unfertilized sea urchin eggs generates an intracellular free calcium concentration ([Ca]) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTPyS-induced transient is blocked by prior microinjection of the inositol trisphosphate (InsP3) antagonist heparin. GTPyS injection also causes stimulation of the egg's Na+/H+ antiporter via protein kinase C, even in the absence of a [Cal, increase. These data suggest that GTPyS acts by stimulating the calciumindependent production of the phosphoinositide messengers InsP3 and diacylglycerol (DAG). However, the fertilization [Ca], transient is not affected by heparin, nor can the sperm cause calcium-independent stimulation of protein kinase C. It seems that the bulk of InsP3 and DAG production at fertilization is triggered by the [Ca], transient, not by the sperm itself. GDP,#S, a G-protein antagonist, does not affect the fertilization [Ca], transient. Our findings do not support the idea that signal transduction at fertilization operates via a G-protein linked directly to a plasma membrane sperm receptor.

Apoptosis, Aug 8, 2013

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathog... more Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyaninderived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

The conversation, 2016

Foundation and The Alliance for Useful Evidence, as well as sixty five university members. Dear c... more Foundation and The Alliance for Useful Evidence, as well as sixty five university members. Dear class of 2016, Finishing school can be a daunting experience but you are young, bright and have your future ahead of you — easy for me to say, you might think. I could fill a book with the things I wish I'd known when I left school – how to iron, how to put up a shelf, how invaluable learning languages is. Though the biggest one is how everyone is in the same boat, and how no one really knows what they want to do when they leave school. So, with this in mind, let me impart some of my age old wisdom onto your young shoulders. Here are some of the most important life lessons I wish somebody had taught me before the final school bell rang.

Journal of Innate Immunity, 2017

Cellular immune defences in sea urchins are shared amongst the coelomocytes - a heterogeneous pop... more Cellular immune defences in sea urchins are shared amongst the coelomocytes - a heterogeneous population of cells residing in the coelomic fluid (blood equivalent) and tissues. The most iconic coelomocyte morphotype is the red spherule cell (or amebocyte), so named due to the abundance of cytoplasmic vesicles containing the naphthoquinone pigment echinochrome A. Despite their identification over a century ago, and evidence of antiseptic properties, little progress has been made in characterising the immunocompetence of these cells. Upon exposure of red spherule cells from sea urchins, i.e., Paracentrotus lividus and Psammechinus miliaris, to microbial ligands, intact microbes, and damage signals, we observed cellular degranulation and increased detection of cell-free echinochrome in the coelomic fluid ex vivo. Treatment of the cells with ionomycin, a calcium-specific ionophore, confirmed that an increase in intracellular levels of Ca2+ is a trigger of echinochrome release. Incubatin...