Ultrastructural localization of carbonic anhydrase in tissues involved in shell formation and ionic regulation in the pond snail Lymnaea stagnalis

The ultrastructural localization of carbonic anhydrase (CA) was investigated with the cobalt-bicarbonate method in three epithelia of the pond snail Lymnaea stagnalis. In the epidermis a selective population of "positive cells" was observed. In these cells, CA is confined to the apical and to small parts of the lateral plasma membrane. In cells of the outer mantle epithelium, CA is localized in the lateral and basal parts of the plasma membrane. In cells of the ureter, CA was found apically as well as basally. The localization of CA is discussed in relation to the different functions of the epidermis (electrolyte uptake), mantle (HCO3- secretion, calcification) and ureter (electrolyte uptake, acid-base regulation).     

Potential for control of signaling pathways via cell size and shape

BACKGROUND: In order for signals generated at the plasma membrane to reach intracellular targets, activated messengers, such as G proteins and phosphoproteins, must diffuse through the cytoplasm. If the deactivators of these messengers, GTPase activating proteins (GAPs) and phosphatases, respectively, are sufficiently active in the cytoplasm, then the signal could in principle decay before reaching the target and a stable spatial gradient in phosphostate would be generated. Recent experiments document the existence of such gradients in living cells and suggest a role for them in mitotic spindle morphogenesis and cell migration. However, how such systems behave theoretically when embedded in a cell of varying size or shape has not been considered. RESULTS: Here we use a simple mathematical model to explore the theoretical consequences of a plasma membrane bound activator (i.e., guanine nucleotide exchange factor, GEF, or kinase) and a cytoplasmic deactivator (i.e., GAP or phosphatase), and we find that as a model cell grows, the substrate becomes progressively dephosphorylated as a result of decreased proximity to the activator. Conversely, as a cell spreads and flattens, the substrate becomes globally phosphorylated because of increased proximity of the substrate to the activator. Similarly, in the leading edge of polarized cells and in protrusions such as lamellipodia or filopodia, the substrate is highly phosphorylated. As a specific test of the model, we found that the experimentally observed preferential activation of the G protein Cdc42 in the periphery of fibroblasts that was recently reported is consistent with model predictions. CONCLUSIONS: We conclude that cell-signaling pathways can theoretically be turned on and off, both locally and globally, in response to alterations in cell size and shape.     

INORGANIC CARBON LIMITATION, EXOFACIAL CARBONIC ANHYDRASE ACTIVITY, AND PLASMA MEMBRANE REDOX ACTIVITY IN MARINE PHYTOPLANKTON SPECIES

In the marine phytoplankton species tested, a possible link between exofacial ferricyanide reduction at the plasma membrane of intact cells, inorganic carbon status of the cells, and extracellular carbonic anhydrase (CA) activity is proposed. In species with no extracellular CA activity under carbon-limited or carbon-replete conditions, barely detectable ferricyanide reduction was observed. Species in which extracellular CA was only detected under carbon-limited conditions showed high rates of exofacial ferricyanide reduction, as shown previously for Skeletonema costatum. Immunological analysis has demonstrated that the CA protein was present in both carbon-limited and carbon-replete cells, even though the CA activity could only be detected when inorganic carbon was limiting. Incubation of the inactive extracellular CA protein from carbon-replete cells with DTT caused activation of the enzyme. It is proposed that CA limitation in the light promotes proton extrusion and increased plasma membrane redox activity, which result in the protonation and activation of the extracellular CA.     

Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells.

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.     

Expression and subcellular localization of a 35-kDa carbonic anhydrase IV in a human pancreatic ductal cell line (Capan-1).

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.     

Substrate- and ubiquitin-dependent trafficking of the yeast siderophore transporter Sit1

Eukaryotic plasma membrane transporters are subjected to a tightly regulated intracellular trafficking. The yeast siderophore iron transporter1 (Sit1) displays substrate-regulated trafficking. It is targeted to the plasma membrane or to a vacuolar degradative pathway when synthesized in the presence or absence of external substrate, respectively. Sorting of Sit1 to the vacuolar pathway is dependent on the clathrin adaptor Gga2, and more specifically on its C-GAT subdomain. Plasma membrane undergoes substrate-induced ubiquitylation dependent on the Rsp5 ubiquitin protein ligase. Sit1 is also ubiquitylated in an Rsp5-dependent manner in internal compartments when expressed in the absence of substrate. In several rsp5 mutants including cells deleted for RSP5 , Sit1 expressed in the absence of substrate is correctly targeted to the endosomal pathway but its sorting to multivesicular bodies (MVBs) is impaired. Consequently, it displays endosome to plasma membrane targeting, with kinetics similar to those observed in vps mutants defective for MVB sorting. Plasma membrane Sit1 is modified by Lys63-linked ubiquitin chains. We also show for the first time in yeast that modification by this latter type of ubiquitin chains is required directly or indirectly for efficient MVB sorting, as it is for efficient internalization at the plasma membrane.     

Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells

The high concentration of HCO₃ ^– ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl^–/HCO₃ ^– exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors (n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH₂ antibody directed against the NH₂ terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.The first two authors have contributed equally to this work     

Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.     

Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts

Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.     

Novel features of amphiphilic peptide Mas7 in signalling via heterotrimeric G-proteins.

Amphiphilic peptide Mas7, a structural analogue of mastoparan is a known activator of heterotrimeric Gi-proteins and its downstream effectors. This study investigated the functional interaction of Mas7 with a plasma membrane protein from CHO cells, the endogenous mono-ADP-ribosyltransferase. The substrate of endogenous mono-ADP-ribosyltransferase was the ADP-ribosylated protein with a molecular mass of 36 kDa, which corresponded to the beta subunit of heterotrimeric G-proteins. The effect of Mas7 on endogenous mono-ADP-ribosyltransferase activity was in the micromolar range with a maximal activation of 205% over the basal. In pertussis treated plasma membranes, it was found that the effect of Mas7 on endogenous mono-ADP-ribosyltransferase was partially blocked, which suggests the involvement of G-proteins, such as Gi or G0. In addition, an immunoassay was developed for the visualization of interaction between the a subunit and the betagamma dimer of G-protein on a Ni-NTA support. The physical interaction was tested of Mas7 with the heterotrimeric G-protein alphai2 subunit, which was overexpressed together with beta1gamma2-His6 subunits in sf9 cells. An interaction between Gi2 heterotrimer and Mas7 was not observed, which was not in accordance with previously reported results of mastoparan obtained for Gi-proteins from bovine brain. In conclusion, the signal is mediated from Mas7 to endogenous mono-ADP-ribosyltransferase via pertussis sensitive G-proteins. Furthermore, it is hypothesized that Gi2 G-proteins are not involved in the process.     

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.     

海洋浮游藻类细胞质膜氧化还原活性与细胞外CA活性的关系

海洋浮游藻类除通过吸收和释放分子与离子来改变其环境的化学成分外,还可通过细胞外表面一些酶的作用引起质膜外化学物质变化。在这方面,海洋浮游藻类一个主要的细胞外表面酶-碳酸酐酶(CA),在经胰蛋白酶处理从细胞质膜上释放出来后,仍保留其催化活性。当细胞外表面CA(简称细胞外CA)具活性时,可催化质膜外HCO_3~-与CO_2的相互转化,为Rubisco(磷酸核酮糖羧化酶)提供一稳定的CO_2流量环境,以维持正常的光合作用。     

大鼠附睾钠钾离子主动转运的超微结构研究

在超微结构水平上研究大鼠附睾的离子转运机制,确定离子主动转运相关的钠-钾三磷酸腺苷酶(Na-K-ATPase)的分布。结果表明大鼠附睾上皮细胞具有二类NPP酶活性,即依赖于钾的,对乌巴因敏感的和对Levamisole有抵抗作用的K-NPP酶;不依赖于钾的,对乌巴因不敏感的和对Levamisole有抵抗作用的非K-NPP酶。K-NPP酶只分布在大鼠附睾各段和输出小管近段的主细胞基底膜与侧膜上,非K-NPP酶分布在主细胞的高尔基氏体、内质网膜和致密体上。附睾上皮细胞中的基底细胞、晕细胞和亮细胞均缺少酶的分布。低钾液附睾组织灌流和用棉酚处理动物,均使非钾-NPP酶活性增强,对K-NPP酶活性有抑制作用。     

海洋浮游藻类细胞质膜氧化还原活性与细胞外CA活性的关系（简报）

Exofacial ferricyanide reduction at the plasma membrane of intact cells, and the link between plasma membrane redox activity, inorganic carbon status of the cells and extracellular carbonic anhydrase (CAext) activity were assayed using 10 marine phytoplankton species. In species Chaetocceros compressus, Cocolithus pelagicus and Gephyrocapsa ocetanica with no extracellular CA activity under carbon-limited or carbon-replete conditions, barely detectable ferricyanide reduction was observed. Species Skeletonema costatum, Melosira sp., Thalassiosira rotula, Thalassiosira weisflogi and Pleurochrysis carterae in which extracellular CA activity was only detected under carbon-limited conditions showed high rates of exofacial ferricyanide reduction. Western blotting and immunolocalization showed the presence of enzyme protein under carbon-limited and replete conditions at the cell surface, even though the CA activity could only detected when inorganic carbon was limiting, which suggests that the development of extracellular CA in response to carbon limitation is an activation of a preexisting protein rather than de novo synthesis. The results suggest that inorganic carbon limitation in the light increases plasma membrane redox activity and promotes proton extrusion, which result in the protonation and activation of the extracellular CA.     

Targeting of carbonic anhydrase IV to plasma membranes is altered in cultured human pancreatic duct cells expressing a mutated (deltaF508) CFTR

Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.     

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.     

Salt-induced cooperativity in ATPase activity of plasma membrane-enriched fractions from cultured Citrus cells: kinetic evidence

ATPase activity was studied in plasma membrane-enriched fractions prepared from cultured Citrus sinensis L. cv. Osbeck cells. In general, properties of the plasma membrane ATPase from cultured cells, such as optimal pH and temperature. V_max and K_m were similar to those already observed in higher plants. The effects of high salt concentrations on ATPase activity were studied in membrane fractions derived from salt-sensitive and salt-tolerant cells grown in the presence or absence of salt. NaCl did not have an in vivo effect on V_max and the apparent K_m value for ATP. However, high concentrations of NaCl, or KCl, added in vitro, induced cooperativity in the enzyme and reduced the affinity of the enzyme for its substrate. Isoosmolar concentrations of sucrose or choline chloride failed to do so. Our results suggest that the plasma membrane ATPase of Citrus cells has more than one substrate-binding site on the native form of the enzyme which interact in the presence of salt and act independently in its absence.     

The Src homology 2-containing inositol 5-phosphatase 1 (SHIP1) is involved in CD32a signaling in human neutrophils

Phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P(3)) plays important signaling roles in immune cells, particularly in the control of activating pathways and of survival. It is formed by a family of phosphatidylinositol 3'-kinases (PI3Ks) which phosphorylate PtdIns(4,5)P(2) in vivo. In human neutrophils, the levels of PtdIns(3,4,5)P(3) increase rapidly at the leading edge of locomoting cells and at the base of the phagocytic cup during FcgammaR-mediated particle ingestion. Even though these, and other, data indicate that PtdIns(3,4,5)P(3) is involved in the control of chemotaxis and phagocytosis in human neutrophils, the mechanisms that regulate its levels have yet to be fully elucidated in these cells. We evaluated the potential implication of SHIP1 and PTEN, two lipid phosphatases that utilize PtdIns(3,4,5)P(3) as substrate, in the signaling pathways called upon in response to CD32a cross-linking. We observed that the cross-linking of CD32a resulted in a transient accumulation of PtdIns(3,4,5)P(3). CD32a cross-linking also induced the tyrosine phosphorylation of SHIP1, its translocation to the plasma membrane and its co-immunoprecipitation with CD32a. CD32a cross-linking had no effect on the level of serine/threonine phosphorylation of PTEN and did not stimulate its translocation to the plasma membrane. PP2, a Src kinase inhibitor, inhibited the tyrosine phosphorylation of SHIP1 as well as its translocation to the plasma membrane. Wortmannin, a PI3K inhibitor, had no effect on either of these two indices of activation of SHIP1. Our results indicate that SHIP1 is involved, in a Src kinase-dependent manner, in the early signaling events observed upon the cross-linking of CD32a in human neutrophils.     

Exogenous basement-membrane-like matrix stimulates adrenergic development in avian neural crest cultures

The development of quail trunk neural crest cultures was dramatically altered when the cultures were overlaid with a gel of reconstituted basement membrane (RBM) components derived from the Engelbreth-Holm-Swarm sarcoma. In the presence of the RBM gel overlay, the number of catecholamine-positive (CA+) cells that developed was increased 50-fold, while the final number of melanocytes and total cells was only half that seen in the control cultures. The presence of the RBM gel overlay did not alter the time of onset of differentiation of the CA+ cells or melanocytes. The stimulation of CA+ cell number was not observed with type IV collagen substrates, laminin substrates or type I collagen gel overlays with or without added laminin. The stimulation of CA+ cell development was dependent on initial plating density. The number of CA+ cells that developed in the presence of the RBM gel was proportional to the initial plating density at 80-320 cells mm-2, whereas no CA+ cells were observed below 20 cells mm-2 and only a few CA+ cells were detected at 40 cells mm-2. There was, however, extensive cell division and differentiation of melanocytes and unpigmented cells at the lower initial plating densities. When the RBM gel was used as a substrate, rather than as an overlay, a striking rearrangement of cells into interconnected strands was observed. After several days in culture, melanocytes, CA+ cells and unpigmented cells were present in these strands. These results indicate that molecules associated with a reconstituted basement-membrane-like matrix are a potent stimulatory influence on adrenergic development and also act to inhibit the production of other cell types in neural crest cultures.