High-throughput screening identifies compounds that enhance lentiviral transduction.

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

A short N-terminal sequence of PTEN controls cytoplasmic localization and is required for suppression of cell growth.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.

Bronchoalveolar fluid is not a major hindrance to virus-mediated gene therapy in cystic fibrosis.

Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency. However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer. We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor. We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors. Non-CF BAL significantly impaired adenovirus-mediated gene transfer. Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency. As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.

Small molecular weight secretory factors from Pseudomonas aeruginosa have opposite effects on IL-8 and RANTES expression by human airway epithelial cells.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.

Inhibition of Ca(2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.

Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca(2+) concentration (¿Ca(2+)(c)) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca(2+) signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in ¿Ca(2+)(c) in human macrophages, no change in ¿Ca(2+)(c) occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased ¿Ca(2+)(c), as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in ¿Ca(2+)(c) similar to COZ. Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca(2+) signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion and promotion of intracellular mycobacterial survival.

rab5 GTPase regulates adenovirus endocytosis.

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

IL-4 and IFN-gamma increase steady state levels of polymeric Ig receptor mRNA in human airway and intestinal epithelial cells.

Delivery of IgA to the mucosal surface occurs via transcytosis of polymeric IgA (pIgA) across the epithelium, a process mediated by the pIgR. Several factors increase pIgR expression in human epithelial cells, including IL-4 and IFN-gamma. Using an RNase protection assay, we found that IL-4 and IFN-gamma increase steady state levels of pIgR mRNA in both human intestinal (HT29) and airway (Calu-3) epithelial cells. Time course studies in HT29 clone 19A cells showed that with each cytokine alone and with both together: 1) there was a significant lag before mRNA levels increased; 2) maximal levels were not reached until 48-72 h after the addition of cytokines; 3) mRNA levels remained elevated in the continued presence of cytokines; and 4) addition of actinomycin D or removal of cytokines led to decreases in mRNA levels with a half-life of approximately 20-28 h. Cytokine-dependent increases in steady state levels of pIgR mRNA were inhibited by cycloheximide and by protein tyrosine kinase inhibitors but not by inhibitors of protein kinase C or cAMP-dependent protein kinase A. Both IFN-gamma and IL-4 increased expression of the inducible transcription factor IFN regulatory factor-1 (IRF-1), but levels of IRF-1 only weakly correlated with levels of pIgR mRNA, suggesting that additional transcription factors are required. These studies provide additional insights into the mechanisms by which cytokines regulate expression of the pIgR, a central player in mucosal immunity.

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.

Pseudomonas pyocyanine alters calcium signaling in human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes both acute and chronic lung disease. P. aeruginosa exerts many of its pathophysiological effects by secreting virulence factors, including pyocyanine, a redox-active compound that increases intracellular oxidant stress. Because oxidant stress has been shown to affect cytosolic Ca2+ concentration ([Ca2+]c) in other cell types, we studied the effect of pyocyanine on [Ca2+]c in human airway epithelial cells (A549 and HBE). At lower concentrations, pyocyanine inhibits inositol 1,4,5-trisphosphate formation and [Ca2+]c increases in response to G protein-coupled receptor agonists. Conversely, at higher concentrations, pyocyanine itself increases [Ca2+]c. The pyocyanine-dependent [Ca2+]c increase appears to be oxidant dependent and to result from increased inositol trisphosphate and release of Ca2+ from intracellular stores. Ca2+ plays a central role in epithelial cell function, including regulation of ion transport, mucus secretion, and ciliary beat frequency. By disrupting Ca2+ homeostasis, pyocyanine could interfere with these critical functions and contribute to the pathophysiological effects observed in Pseudomonas-associated lung disease.

Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures.

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.

cAMP and inositol 1,4,5-trisphosphate increase Ca2+ in HT-29 cells by activating different Ca2+ influx pathways.

Ca2+ plays a central role in regulating transepithelial fluid and electrolyte transport in intestinal epithelial cells. To investigate the mechanisms regulating the cytosolic free Ca2+ concentration ([Ca2+]c), we examined the effect of secretory agonists on [Ca2+]c in the intestinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2+]c increased after addition of either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P3, whereas carbachol increased cellular levels of Ins(1,4,5)P3 and stimulated inositol phosphate turnover without increasing cAMP. Second, carbachol increased [Ca2+]c by stimulating the release of Ca2+ from intracellular stores and influx of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]c by stimulating Ca2+ influx alone. Third, the responses to maximal concentrations of cAMP agonists and carbachol were approximately additive. Finally, Ins(1,4,5)P3- but not cAMP agonist-dependent Ca2+ influx was inhibited by inorganic Ca2+ channel blockers. Thus, in intestinal epithelial cells, [Ca2+]c is regulated by at least two different second-messenger pathways, involving Ins(1,4,5)P3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P3.

Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells.

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl-) transport. One possible pathway for Cl- secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl- channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl- secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (psi t) = -1.1 +/- 0.1 mV, transepithelial resistance (Rt) = 768 +/- 58 omega.cm2, and short-circuit current (Isc) = 1.9 +/- 0.2 microA/cm2 (mean +/- SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl- channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Isc, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysfunction of CFTR bearing the delta F508 mutation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.

Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes.

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.

Purification and characterization of clathrin-coated vesicles from Chlamydomonas.

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.