Permeabilized Cell Models Research Articles

Supramolecular delivery of dinuclear ruthenium and osmium MCU inhibitors.

The transmembrane protein known as the mitochondrial calcium uniporter (MCU) mediates the influx of calcium ions (Ca2+) into the mitochondrial matrix. An overload of mitochondrial Ca2+ ( m Ca2+) is directly linked to damaging effects in pathological conditions. Therefore, inhibitors of the MCU are important chemical biology tools and therapeutic agents. Here, two new analogues of previously reported Ru- and Os-based MCU inhibitors Ru265 and Os245, of the general formula [(C10H15CO2)M(NH3)4(μ-N)M(NH3)4(O2CC10H15)](CF3SO3)3, where M = Ru (1) or Os (2), are reported. These analogues bear adamantane functional groups, which were installed to act as guests for the host molecule cucurbit-[7]-uril (CB[7]). These complexes were characterized and analyzed for their efficiency as guests for CB[7]. As shown through a variety of spectroscopic techniques, each adamantane ligand is encapsulated into one CB[7], affording a supramolecular complex of 1 : 2 stoichiometry. The biological effects of these compounds in the presence and absence of two equiv. CB[7] were assessed. Both complexes 1 and 2 exhibit enhanced cellular uptake compared to the parent compounds Ru265 and Os245, and their uptake is increased further in the presence of CB[7]. Compared to Ru265 and Os245, 1 and 2 are less potent as m Ca2+ uptake inhibitors in permeabilized cell models. However, in intact cell systems, 1 and 2 inhibit the MCU at concentrations as low as 1 μM, marking an advantage over Ru265 and Os245 which require an order of magnitude higher doses for similar biological effects. The presence of CB[7] did not affect the inhibitory properties of 1 and 2. Experiments in primary cortical neurons showed that 1 and 2 can elicit protective effects against oxygen-glucose deprivation at lower doses than those required for Ru265 or Os245. At low concentrations, the protective effects of 1 were modulated by CB[7], suggesting that supramolecular complex formation can play a role in these biological conditions. The in vivo biocompatibility of 1 was investigated in mice. The intraperitoneal administration of these compounds and their CB[7] complexes led to time-dependent induction of seizures with no protective effects elicited by CB[7]. This work demonstrates the potential for supramolecular interactions in the development of MCU inhibitors.

Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury

Novel therapeutic strategies to treat mitochondrial deficiencies in acute coronary syndromes are needed. Complex I of the mitochondrial electron transport system is damaged following ischemia/reperfusion (I/R) injury. This disruption contributes to aberrant electron transport, diminished bioenergetics, an altered redox environment, and mitochondrial damage involved in tissue injury. In this study, we determined the cardiac and mitochondrial effects of idebenone, a benzoquinone currently in several clinical trials with purported ‘antioxidant’ effects. We employed complimentary models of ischemia/reperfusion injury in perfused hearts, permeabilized cardiac fibers, isolated mitochondria, and in cells to elucidate idebenone's cardioprotective mechanism(s). In ex vivo whole hearts, infarct size was markedly reduced with post-ischemic idebenone treatment (25 ± 5% area at risk, AAR) compared to controls (56 ± 6% AAR, P < .05). Several parameters of hemodynamic function were also significantly improved after idebenone treatment. Parallel studies of anoxia/reoxygenation were conducted using isolated mitochondria and permeabilized ventricular fibers. In isolated mitochondria, we simultaneously monitored respiration and ROS emission. Idebenone treatment modestly elevated succinate-derived H2O2 production when compared to vehicle control (1.34 ± 0.05 vs 1.21 ± 0.05%, H2O2/O2 respectively, P < .05). Isolated mitochondria subjected to anoxia/reoxygenation demonstrated higher rates of respiration with idebenone treatment (2360 ± 69 pmol/s*mg) versus vehicle control (1995 ± 101 pmol/s*mg). Both mitochondria and permeabilized cardiac fibers produced high rates of H2O2 after anoxia/reoxygenation, with idebenone showing no discernable attenuation on H2O2 production. These insights were further investigated with studies in mitochondria isolated from reperfused ventricle. The profound decrease in complex-I dependent respiration after ischemia/reperfusion (701 ± 59 pmolO2/s*mg compared to 1816 ± 105 pmol O2/s*mg in normoxic mitochondria) was attenuated with idebenone treatment (994 ± 76 vs pmol O2/s*mg, P < .05). Finally, the effects of idebenone were determined using permeabilized cell models with chemical inhibition of complex I. ADP-dependent oxidative phosphorylation capacity was significantly higher in complex-I inhibited cells treated acutely with idebenone (89.0 ± 4.2 pmol/s*million cells versus 70.1 ± 8.2 pmol/s*million cells in untreated cells). Taken together, these data indicate that the cardioprotective effects of idebenone treatment do not involve ROS-scavenging but appear to involve augmentation of the quinone pool, thus providing reducing equivalents downstream of complex I. As this compound is already in clinical trials for other indications, it may provide a safe and useful approach to mitigate ischemia/reperfusion injury in patients.

Differential localization and functional specialization of centrin analogs in the parasitic ciliate Trichodina pediculus.

Trichodinids are ciliated protozoans that reversibly attach to the tegument of marine and freshwater host-organisms via an adhesive disc. In this study, we have used permeabilized cell models of Trichodina pediculus to examine the distribution of centrins, a Ca(2+)-binding protein associated with centrioles and/or contractile filamentous structures in a large number of protists. The previous finding that filamentous material of the adhesive disc comprised a 23-kDa centrin analog suggested that this protein might be a disc-specific isoform. This possibility was explored through immunolabeling methods using two distinct antibodies, anti-ecto-endoplasmic boundary (EEB) and anti-Hscen2 previously shown to react respectively with centrin-based filament networks and with centrioles. Immunofluorescence microscopy showed that anti-EEB reacts with filamentous material of the disc but not with basal bodies. Conversely, anti-Hscen2 cross-reacted with basal bodies but failed to label any type of structure occurring in the disc area. More detailed data on localization of this protein was obtained by immunoelectron microscopy showing gold particles deposits in the lumen of basal bodies. The different patterns revealed by this immunochemical approach suggest that the two protein antigens concerned by this study are distinct centrin isoforms that presumably perform organelle-specific function in the ciliate T. pediculus.

Differential modulation of mu‐opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent

Regulators of G protein signaling (RGS) proteins act as GTPase-accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase by [D-Ala(2),N-Me-Phe(4),Gly-ol]-enkephalin (DAMGO), a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Galphai2 and minimal GTPase-accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.

Regulators of G protein signaling attenuate opioid inhibition of adenylyl cyclase in a permeabilized C6 cell model

Regulators of G protein signaling (RGS) proteins terminate G protein signaling by accelerating the rate of GTP hydrolysis of Gα subunits. Recent evidence implicates RGS proteins as key players in shaping the response to both acute and chronic opioids, but the relative contribution of individual RGS proteins in these processes is poorly understood. In order to determine the effects of specific, purified RGS proteins on opioid signaling C6 cells stably expressing the mu opioid receptor were rendered permeable to ions and proteins by treatment with digitonin. Forskolin stimulation of adenylyl cyclase (AC) was equipotent in permeabilized vs. non-permeabilized cells. DAMGO, a mu-specific opioid peptide, inhibited forskolin-sitmulated AC activity with slightly reduced potency in permeabilized cells (18±1.7 nM) compared to non-permeabilized cells (4.0±1.3 nM) but was equally efficacious under either condition, indicating that opioid signaling to AC remained fully intact under permeabilization conditions. Purified RGS4 box domain added to permeabilized cells resulted in as much as a two-fold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. Also, the inhibitory effect of DAMGO was reduced to the same extent and in a non-additive manner by full length RGS4 and RGS8. In contrast, RGS7 box had no effect. These data suggest that specific RGS proteins differentially regulate opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling. Supported by DA04087, GM39561, T32 DA007268

Augmented Ciliary Reorientation Response and cAMP-dependent Protein Phosphorylation Induced by Glycerol in Triton-extracted Paramecium

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10(-6) M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orientation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.

Reactivation of Ca2+-dependent cytoplasmic contraction in permeabilized cell models of the heliozoon Echinosphaerium akamae.

Permeabilized cell models of the large heliozoon Echinosphaerium akamae were prepared by treatment with 100 mM EGTA or 1% Triton X-100. When > 10(-6) M Ca(2+) was added to the EGTA-permeabilized cells, axopodial cytoplasm became contracted and several swellings were formed along the axopodial length. Axonemal microtubules remained intact, while higher concentration of Ca(2+) (> 10(-4) M) induced microtubule disassembly and complete breakdown of the axopodia. In Triton-permeabilized cells, cytoplasmic contraction and relaxation of the cell body were induced repeatedly by successive addition and removal of Ca(2+). The contraction did not require ATP, and was not inhibited by cytochalasin B. Electron microscopy showed, in EGTA-permeabilized axopodia, contractile tubules became granulated by the addition of Ca(2+). From these observations, it is strongly suggested that Ca(2+)-dependent granulation of the contractile tubules is responsible for the axopodial contraction.

Ca 2+-dependent cytoplasmic contractility of the heliozoon Actinophrys sol

A “contractile tubules structure (CTS)” has been described in the radiating axopodia and cytoplasm of the heliozoon Actinophrys sol. Permeabilized cell models with fully-extended axopodia were prepared with 100 mM EGTA. When Ca2+ was added to the permeabilized cells, cytoplasmic swellings formed along the length of axopodia in a concentration-dependent manner. Triton X-100 was also used to prepare cell models of heliozoa. In the Triton-extracted cell model, axopodia were detached, but the cell body remained as a mass of cytoplasm which showed repetitive contraction and relaxation in a Ca2+-dependent manner with 10–8 M as its threshold level. A cell homogenate of A. sol was found to yield a precipitate following the addition of Ca2+. Formation of the precipitate occurred within a few minutes after the addition of Ca2+, and was dependent on the concentration of Ca2+. Electron microscopy showed that the CTS-like filamentous structures were present in the cell homogenate, and that the precipitate obtained from the cell homogenate by adding Ca2+ was composed of granular aggregates morphologically similar to the transformed CTS observed after axopodial contraction.

α‐granule secretion from α‐toxin permeabilized, MgATP‐exposed platelets is induced independently by H and Ca2

In order to better understand granule release from platelets, we developed an α-toxin permeabilized platelet model to study α-granule secretion. Secretion of α-granules was analyzed by flow cytometry using P-selectin as a marker for α-granule release. P-selectin surface expression occurred when platelets were permeabilized in the presence of Ca2+. Responsiveness to Ca2+ was lost 30 min after permeabilization but could be reconstituted with MgATP. α-toxin–permeabilized, MgATP-exposed platelets also degranulated within a pH range of 5.4–5.9 without exposure to and independent of Ca2+. ATP, GTP, CTP, UTP, and ITP supported Ca2+-induced α-granule secretion, while H+-induced α-granule secretion occurred only with ATP and GTP. Both Ca2+- and H+-induced α-granule secretion required ATP hydrolysis. Kinase inhibitors blocked both Ca2+- and H+-induced secretion. These data suggest that α-granule secretion in this permeabilized platelet system shares many characteristics with granule secretion studied in other permeabilized cell models. Furthermore, these results show that H+ can trigger α-granule release independent of Ca2+. J. Cell. Physiol. 179:1–10, 1999. © 1999 Wiley-Liss, Inc.

Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER-mitochondrial Ca2+ coupling and synaptic transmission.

Ionic Control of Chromosome Architecture in Living and Permeabilized Cells

Studies with isolated chromatin show that higher order chromosome architecture can be regulated by ionic conditions; however, the physiological relevance of these findings remains unknown. In the present study, chromosome architecture was analyzedin situin living and detergent-extracted cells exposed to different ionic conditions. In intact mitotic endothelial cells, chromosomes instantly unfolded as detected by phase contrast microscopy when the salt concentration in the culture medium was increased from 110 to 410 mM NaCl or from 0 to 65 mM MgCl2. When the ions were removed and the preexisting culture conditions were restored, chromosomes refolded into their original shapes and subsequently underwent mitotic division. Similar reversible effects were observed on nucleolar structure in living interphase cells as well as on mitotic chromosomes exposed to high salt after cell membranes were removed by treatment with Triton X-100. This permeabilized mitotic cell model was then used to identify proteins that remained tightly associated with chromatin during the ion-driven chromosome unfolding–refolding cycle and which therefore could be important for maintenance of chromosome structure. Under these conditions in which disassembled chromosomes retained their ability to fully recondense, more than 95% of Topoisomerase I was extracted whereas approximately 25% of Topoisomerase IIα and 50% of Histone H1 remained tightly associated with chromatin. These data demonstrate the sensitivity of chromosome structure to variations in ionic concentrationin situand suggest that there are at least two distinct pools of Histone H1 and Topoisomerase IIα associated with chromatin during mitosis, one of which may be required for chromosome compaction.

Cell surface and organelle transport share the same enzymatic properties in Reticulomyxa.

Reticulomyxa transports particulates, like bacterial and algal prey items, bidirectionally along the outside of its pseudopodia. This cell surface transport and intracellular organelle transport can be reactivated in detergent permeabilized cell models [Orokos et al., 1997: Cell Motil. Cytoskeleton]. We have used this unique system to compare cell surface and organelle mechanochemistry in situ in the same reactivated pseudopodia. The ATPase activities of both types of transport were indistinguishable; each displayed identical nucleoside triphosphate specificity, transport ATPase kinetics, and inhibitor sensitivity. Organelle and cell surface transport reactivation required "hydrolyzable" adenosine nucleoside triphosphates; neither reactivated with GTP, CTP, UTP, ITP, AMP-PNP, AMP-PCP, or ATP-gamma-S. However, other ATP analogues, such as 2'-deoxy-ATP and 3'-deoxy-ATP and 2',3'-dideoxy-ATP, supported the reactivation of organelle and cell surface transport at similar, but markedly reduced, velocities. Both transport processes were inhibited similarly by known inhibitors of dynein ATPases such as erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) or sodium (Na)-orthovanadate. N-ethylmaleimide (NEM) and ultraviolet (UV) irradiation in the presence of Na-orthovanadate and ATP permanently disabled both transport processes. Organelle and surface transport followed identical Michaelis-Menten kinetics with a calculated Km of 118 microM ATP and a maximum translocation velocity (Vmax) of 8.33 microm/sec. These findings strongly suggest that cell surface transport shares the same cytoplasmic dynein motor [Schliwa et al., 1991: J. Cell Biol. 112:1199-1203] that drives organelle transport.

Cell surface and organelle transport share the same enzymatic properties in Reticulomyxa

Reticulomyxa transports particulates, like bacterial and algal prey items, bidirectionally along the outside of its pseudopodia. This cell surface transport and intracellular organelle transport can be reactivated in detergent permeabilized cell models [Orokos et al., 1997: Cell Motil. Cytoskeleton]. We have used this unique system to compare cell surface and organelle mechanochemistry in situ in the same reactivated pseudopodia. The ATPase activities of both types of transport were indistinguishable; each displayed identical nucleoside triphosphate specificity, transport ATPase kinetics, and inhibitor sensitivity. Organelle and cell surface transport reactivation required “hydrolyzable” adenosine nucleoside triphosphates; neither reactivated with GTP, CTP, UTP, ITP, AMP-PNP, AMP-PCP, or ATP-γ-S. However, other ATP analogues, such as 2′-deoxy-ATP and 3′-deoxy-ATP and 2′,3′-dideoxy-ATP, supported the reactivation of organelle and cell surface transport at similar, but markedly reduced, velocities. Both transport processes were inhibited similarly by known inhibitors of dynein ATPases such as erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) or sodium (Na)-orthovanadate. N-ethylmaleimide (NEM) and ultraviolet (UV) irradiation in the presence of Na-orthovanadate and ATP permanently disabled both transport processes. Organelle and surface transport followed identical Michaelis-Menten kinetics with a calculated Km of 118 μM ATP and a maximum translocation velocity (Vmax) of 8.33 μm/sec. These findings strongly suggest that cell surface transport shares the same cytoplasmic dynein motor [Schliwa et al., 1991: J. Cell Biol. 112:1199–1203] that drives organelle transport. Cell Motil. Cytoskeleton 38:270–277, 1997. © 1997 Wiley-Liss, Inc.

Light-induced chloroplast movements in Lemna trisulca. Identification of the motile system

In mesophyll cells of the water plant Lemna trisulca L. chloroplasts redistribute in response to blue light. In the present study it is shown that an actin depolymerizing agent cytochalasin D, a crosslinker of actin subunits in F-actin m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) as well as N-ethylmaleimide (NEM)—a sulfhydryl group reagent, are potent inhibitors of these blue light-induced chloroplast movements in Lemna. Extraction with cold, buffered glycerol solution preserves light-induced chloroplast arrangements within cells producing permeabilized cell models. ‘Reactivation’ of these cell models by Mg-ATP results in remarkable movements which can be inhibited by treatment with NEM and cytochalasin D. Immunofluorescence microscopy demonstrates that a component which is associated with isolated Lemna chloroplasts cross-reacts with antibodies directed against bovine myosin. These results indicate that a contractile actomyosin system is involved in blue light-induced chloroplast movements in Lemna and a putative motor protein, similar to myosin, is associated with the surface of Lemna chloroplasts.

Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.

PLD AUGMENTS CALCIUM INDUCED DEGRANULATION IN STREPTOLYSIN O PERMEABILIZED NEUTROPHILS. • 901

Neutrophil degranulation is characterized by the fusion of azurophil and specific granules with the plasma membrane. This process is preceded by a complex series of alterations in membrane phospholipid composition mediated, at least in part, by phospholipases C and D. The activation of phospholipase C activation initiates a rise in intracellular Ca2+, while phospholipase D (PLD) hydrolyzes membrane phosphatidylcholine (PC) to phosphatidic acid(PA). (This conversion of PC to PA converts a nonfusogenic phospholipid to a fusogenic one). Using a Streptolysin O permeabilized cell model, we examined the potential role of PLD metabolism on Ca2+-induced degranulation in human neutrophils. Neutrophils permeabilized with streptolysin O can be induced to release their granular contents in response to elevated extracellular [Ca2+]. We found that the addition of exogenous(Streptomyces chromofuscus) PLD caused a dose-dependent augmentation in the Ca2+-induced release of both specific (lactoferrin) as well as azurophil (β-glucuronidase) granular contents (Table 1). Secretion was dependent on both permeabilization as well as the preactivation of PLD with non-stimulatory [Ca2+], indicating that actual metabolism by PLD was important and that it was targeting an intracellular membrane. This work was funded by NICHD and NIAID grants HD28820 and AI20065

Activation of cytosolic phospholipase A 2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A 2 (PLA 2), a 14 kDa ‘secretory’ Type II PLA 2 (sPLA 2) and an 85 kDa ‘cytosolic’ PLA 2 (cPLA 2), that differ in a number of key characteristics: (1) cPLA 2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA 2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA 2 is active at nM calcium (Ca 2+) concentrations; sPLA 2 requires μM Ca 2+ levels. (3) cPLA 2 activity is regulated by phosphorylation; sPLA 2 lacks phosphorylation sites. (4) cPLA 2 is insensitive to reduction; sPLA 2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA 2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [ 3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca 2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [ 3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA 2 activation. Dithiothreitol treatment had little affect on [ 3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA 2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA 2. These results strongly suggested that cPLA 2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA 2 in PMN.

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly.

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.

Translocation of nucleophosmin from nucleoli to nucleoplasm requires ATP

The movement of nucleophosmin from nucleoli to nucleoplasm in HeLa cells induced by cytotoxic drugs and detected by immunofluorescence is inhibited by concomitant treatment with antimycin A in glucose-free medium. Incubation of HeLa cells with antimycin A (300 nM; 30 min) and glucose-free medium resulted in an approximately 90% decrease in cellular ATP pools. To study the biochemical events involved in nucleophosmin translocation, we used an in vitro system consisting of Triton-permeabilized HeLA cells. Incubation of permeabilized cells with ATP (0.5 mM; 1 h) resulted in the translocation of nucleophosmin from nucleoli to nucleoplasm and cytoplasm. Similarly to drug-induced nucleophosmin translocation in whole cultured cells, there is no reduction (measured by e.l.i.s.a.) or degradation of nucleophosmin or change in the ratio of the high-molecular-mass form to the monomeric form (ascertained by Western blotting) during ATP treatment of permeabilized cells. Together, these results indicate a requirement for ATP for redistribution of nucleophosmin from nucleoli to nucleoplasm. Because this permeabilized cell model is simple and efficient and works effectively with exogenous factors, it should provide a powerful tool for investigating the biochemical features of nucleophosmin translocation from nucleoli to nucleoplasm.

The role of actin filaments in the organization of the endoplasmic reticulum in honeybee photoreceptor cells

Close to the bases of the photoreceptive microvilli, arthropod photoreceptors contain a dense network of endoplasmic reticulum that is involved in the regulation of the intracellular calcium concentration, and in the biogenesis of the photoreceptive membrane. Here, we examine the role of the cytoskeleton in organizing this submicrovillar endoplasmic reticulum in honeybee photoreceptors. Immunofluorescence microscopy of taxol-stabilized specimens, and electron-microscopic examination of high-pressure frozen, freeze-substituted retinae demonstrate that the submicrovillar cytoplasm lacks microtubules. The submicrovillar region contains a conspicuous F-actin system that codistributes with the submicrovillar endoplasmic reticulum. Incubation of retinal tissue with cytochalasin B leads to depolymerization of the submicrovillar F-actin system, and to disorganization and disintegration of the submicrovillar endoplasmic reticulum, indicating that an intact F-actin cytoskeleton is required to maintain the architecture of this domain of the endoplasmic reticulum. We have also developed a permeabilized cell model in order to study the physiological requirements for the interaction of the endoplasmic reticulum with actin filaments. The association of submicrovillar endoplasmic reticulum with actin filaments appears to be independent of ATP, Ca2+ and Mg2+, suggesting a tight static anchorage.