Calcium-dependent potassium current in barnacle photoreceptor

When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays to reach a steady value approximately 1 min after depolarization began. The increase in current to the maximum at 4-6s from the minimum at approximately 500 ms is termed the "late current." It is maximum for depolarizations to around +25 mV and is reduced in amplitude at more positive potentials. It is not observed when the membrane is depolarized to potentials more positive than +60 mV. The late current is inhibited by external cobaltous ion and external tetraethylammonium ion, and shows a requirement for external calcium ion. When the calcium-sequestering agent EGTA is injected, the late current is abolished. Illumination of a cell under voltage clamp reduces the amplitude of the late current recorded subsequently in the dark. On the basis of the voltage dependence and pharmacology of the late current, it is proposed that the current is a calcium-dependent potassium current.     

Calcium-dependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14

MRP-8 and MRP-14 are calcium-binding proteins belonging to the S-100 protein family which have been shown to be associated with specific stages of myeloic/monocytic cell differentiation. Members of this protein family are shown to form homo- and heterodimers. Complex formation has also been observed in preliminary experiments for MRP-8 and MRP-14. To evaluate the in vivo relevance of the MRP complex formation and the stoichiometric ratio of individual components complexes were isolated from granulocytes and monocytes by immunoaffinity chromatography using monospecific antibodies. The purified fraction of the MRPs was found to contain monomers and dimers as shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining and immunoblotting. Similar results were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of crude cell extracts. The existence of the MRP complexes in vivo was demonstrated by chemical cross-linking and subsequent isolation of complexes by immunoaffinity chromatography. Two new, highly abundant complexes were found in addition to the heterodimer, but neither monomers nor homodimers were detected. The two larger protein complexes (35.0 and 48.5 kDa) were identified as [MRP-8)2.(MRP-14] trimer and [MRP-8)2.(MRP-14)2) tetramer, respectively. All complexes could be shown to be noncovalently associated in vivo. Furthermore, the association of MRPs was shown to be Ca2+ dependent.     

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells

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Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Minireview: Calcium-dependent proteolysis in living cells

Calcium ion, one of the second messengers in living organisms, has various functions including the ability to enhance intracellular proteolysis. This calcium-dependent proteolysis occurs in the cytosol or membrane rather than in the lysosome. Its mode of action is very wide, including cleavage of hormone receptors, activation of regulatory enzymes and limited proteolysis of the cytoskeletal structure. Although contradictory, the biochemical evidence implies a specified regulatory function of it in the cell. The activation mechanism of a purified calcium-dependent proteinase ( EC 3.4.22.- ) is also discussed.     

Calcium-dependent activation of mitochondrial metabolism in mammalian cells

Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca2+-sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca2+ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH3) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca2+ concentrations and inhibited by higher (microM range) concentrations of the cation. A 2-3-fold stimulation of the activity in response to Ca2+ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca2+ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca2+ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca2+ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca2+ X Mg2+ concentrations in the incubation also influenced the Ca2+ concentration dependence of adenylate cyclase; at high Mg2+ more Ca2+ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH3 cells only in the presence of Ca2+; as Ca2+ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca2+ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH3 cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca2+. Increasing free Ca2+ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca²⁺-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised ¹²⁵I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of ¹²⁵I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered ¹²⁵I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered ¹²⁵I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 m m for ¹²⁵I-transferrin and 1.0 m m for catecholamine, and the intracellular concentrations were 0.1 μ m and 1 μ m , respectively. There was significant ¹²⁵I-transferrin recycling in the virtual absence of intracellular Ca²⁺, but the rate increased when Ca²⁺ was raised above 1 n m , and peaked at 1 μ m when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.     

Calcium-dependent zinc efflux in human red blood cells

Summary Zinc efflux from human red blood cells is largely brought about by a saturable mechanism that depends upon extracellular Ca²⁺ ions. It has aV _max of about 35 mol/10¹³ cells hr, aK _m for external Ca²⁺ of 1×10^–4 m, and aK _m for internal Zn²⁺ of 1×10^–9 m. External Zn²⁺ inhibits with aK _0.5 of 3×10^–6 m. Sr²⁺ is a substitute for external Ca²⁺, but changes in monovalent anions or cations have little effect on the Zn²⁺ efflux mechanism. It is unaffected by most inhibitors of red cell transport systems, although amiloride and D-600 (methoxyverapamil, a Ca²⁺ channel blocker) are weakly inhibitory. The transport is capable of bringing about the net efflux of Zn²⁺, against an electrochemical gradient, provided Ca²⁺ is present externally. This suggests it may be a Zn²⁺:Ca²⁺ exchange, which would be able to catalyze the uphill movement of Zn²⁺ at the expense of an inward Ca²⁺ gradient, which is it self maintained by the Ca²⁺ pump.     

The pigment system of the photoreceptor 7 yellow in the fly,a complex photoreceptor

Summary The 7y photoreceptor in the fly (Musca, Calliphora) retina harbours an unusually complex pigment system consisting of a bistable visual pigment (xanthopsin, X and metaxanthopsin, M), a blue-absorbing C₄₀-carotenoid (zeaxanthin and/or lutein) and a uv sensitizing pigment (3-OH retinol).The difference spectrum and photoequilibrium spectrum in single 7y rhabdomeres were determined microspectrophotometrically (Fig. 2).The extinction spectrum of the C₄₀-carotenoid has a pronounced vibrational structure, with peaks at 430, 450 and 480 nm (Fig. 3). The off-axis spectral sensitivity, determined electrophysiologically with 1 nm resolution shows no trace of this fine structure thus excluding the possibility that the C₄₀-carotenoid is a second sensitizing pigment (Fig. 4).The absorption spectra of X and M are derived by fitting nomogram spectra (based on fly R1–6 xanthopsin) to the difference spectrum. max for X is 425 nm, and for M 510 nm (Fig. 5). It is shown that the photoequilibrium spectrum and the difference spectrum can be used to derive the relative photosensitivity spectra of X and M using the analytical method developed by Stavenga (1975). The result (Fig. 6) shows a pronounced uv sensitivity for both, X and M, indicating that the uv sensitizing pigment transfers energy to both X and M. A value of 0.7 for, the relative efficiency of photoconversion for X and M, is obtained by fitting the analytically derived relative photosensitivity spectra to the absorption spectra at wavelengths beyond 420 nm.     

Deregulation of cyclin E in human cells interferes with prereplication complex assembly

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2-7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E-deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E-mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.     

Presynaptic tubular structures in photoreceptor cells

Summary After the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rat's retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health, U.S.A., (5 RO1 NS 06953-05 NEUA).We want to express our gratitude to Mrs. Haydée Agoff de Zimman and Mr. Alberto Saénz for their skillful technical assistance.     

Properties of barnacle photoreceptor cells in culture

1. Properties of median photoreceptor cells in cultured ocelli from the giant barnacle (Balanus nubilus) were compared in isolated ocelli, ocelli maintained with the supraesophageal ganglion, and fresh ocelli. 2. Cultured photoreceptor cells exhibited slight deterioration after 2-4 weeks. Cell bodies maintained their structure but apparently lost some dendrites. Electron micrographs revealed fewer rhabdomeres. Axons did not degenerate. 3. Intracellularly recorded responses to light in both cultured preparations were qualitatively normal with a small decrease in sensitivity and increase in input resistance. The waveforms of the light responses were normal. 4. The characteristic shadow reflex was maintained after 6 weeks.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.