Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.     

Absence of trypsinogen autoactivation and immunolocalization of pancreatic secretory trypsin inhibitor in acinar cells in vitro

Summary To establish the significance of the addition of trypsin inhibitors to pancreatic acinar cells maintained in vitro, cells were cultured in the presence or absence of soybean trypsin inhibitor. Both cultures exhibited similar growth pattern, ultrastructural appearance, as well as secretory properties. Moreover, there was no evidence of trypsinogen activation in the culture medium. Using the immunocytochemical approach, pancreatic secretory trypsin inhibitor antigenic sites were revealed with specific polyclonal and monoclonal antibodies. The results obtained demonstrated that this trypsin inhibitor is in fact a typical pancreatic secretory protein being processed through the endoplasmic reticulum-Golgi-granule secretory pathway of the acinar cells in rat and human tissues. While the polyclonal antibody yield labelings of increasing intensities along the secretory pathway, the monoclonal one probably due to the molecular nature of its specific antigenic determinant, gave higher labelings in the endoplasmic reticulum. In conclusion the present study has shown that pancreatic acinar cells secrete a specific pancreatic trypsin inhibitor which most probably is involved in the mechanism to prevent trypsinogen activation.     

Identification and immunolocalization of cytoplasmic dynein in Dictyostelium

A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules, UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium. These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.     

Ultrastructural immunolocalization of basic fibroblast growth factor in endothelial cells: morphologic evidence for unconventional secretion of a novel protein

Basic fibroblast growth factor (bFGF) is one of the most potent angiogenic factors. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion. Recent studies suggest that there is an unconventional secretory pathway for this growth factor. The aim of this study was to identify the specific location of bFGF in endothelial cells and to find morphologic evidences concerning its synthesis, storage and release from endothelial cells. The capillaries in hippocampus, adrenal gland, kidney, peripheral nerves as well as the vessels in connective tissues were analysed by using immunogold labeling techniques at electron microscope level. Results show that endogenous bFGF is mainly located in the nuclei of endothelial cells. Slight immunoreactivity is found in the cytoplasm. Immunolabeling is notably absent in pinocytotic vesicles, Golgi complexes, endoplasmic reticulum, nuclear membrane and intercellular junctions. These results provide morphologic evidence suggesting that endothelial cells might export bFGF via unique cellular pathways that are clearly distinct from classical signal peptide mediated secretion and/or release of this protein could be directly through mechanically induced disruptions of these cells. The current study support the recent hypothesis related with unconventional secretory pathway for bFGF as some other “cargo” proteins.     

Identification of an N-domain histidine essential for chaperone function in calreticulin

Calreticulin is an endoplasmic reticulum (ER) luminal Ca(2+)-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt(-/-) cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.     

Calreticulin in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of calreticulin mRNA and protein.

Ca2+ is an essential second messenger for T cell activation, but the exact mechanisms of its action are poorly understood. The cytosolic Ca2+ concentration is significantly increased upon the stimulation of T cells with either mitogen, cross-linking antibodies, or their cognate ligands. In this study, expression of calreticulin, a major Ca(2+)-binding (storage), KDEL protein of the endoplasmic reticulum was examined in resting and concanavalin A (ConA)-stimulated mouse and human T-lymphocytes. Both resting, mouse and human lymphocytes contain very low levels of calreticulin mRNA and protein. Mouse splenocytes stimulated with ConA exhibited an induction in calreticulin mRNA which peaked by Day 4. A 5-fold increase in the immunoreactive calreticulin protein band was also observed in the cells during this period of stimulation. Similarly when human lymphocytes were cultured with ConA a significant increase in the levels of the calreticulin mRNA and protein was observed. The peak of calreticulin mRNA was observed at Day 1 rather than Day 4 as seen for the mouse. These results clearly demonstrate the presence of calreticulin, a Ca(2+)-binding protein originally characterized in muscle tissue, in activated T-lymphocytes. Furthermore, we show that expression of calreticulin correlates with T-lymphocyte activation. Our results suggest that calreticulin may be involved in the signaling pathway for the induction of Ca(2+)-dependent processes and may represent one regulatory mechanism operating in activation of T-lymphocytes.     

Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.     

Identification and immunolocalization of the laminin binding protein from embryonic avian corneal epithelial cells

We report the identification of a 65-kDa laminin-binding protein (LBP) on the basal cell surface of embryonic corneal epithelium in chicken. The 65-kDa LBP was isolated by affinity chromatography with laminin-Sepharose. When reconstituted with lipid vesicles, it demonstrated specific binding for laminin. We produced monoclonal antibodies (MAbs) against 65-k Da LBP; these MAbs immunohistochemically localized to the basal epithelial cell surface. One MAb interfered with the binding of laminin to isolated epithelia and purified 65-k Da LBP. It appears that we have identified, at least in part, a cell-surface binding site for laminin. This site would provide the important link between the extracellular laminin and the intracellular cytoskeleton, and potentially the metabolic machinery of the corneal epithelial cell.     

Immunocytochemistry of calciosomes in liver and pancreas

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3- [2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.     

Identification and characterization of receptors for secretagogues on pancreatic acinar cells

Acinar cells from guinea pig pancreas possess six different classes of receptors that mediate the actions of various secretagogues on enzyme secretion. Four classes of receptors stimulate enzyme secretion by causing mobilization of adenylate cyclase and increased cellular cyclic AMP. This paper summarizes the results of studies that have employed radiolabeled secretagogues of high specific activity and have measured directly the interaction of secretagogues with their receptors on pancreatic acinar cells.     

Huntington disease: no evidence for locus heterogeneity

A total of 63 families with Huntington disease (HD) were examined for linkage between HD and G8 (D4S10). The families included 57 Caucasian, four Black American, and two Japanese. The combined maximum lod score was 87.69 at theta = 0.04 (99% confidence interval 0.018-0.071). The maximum frequency of recombination was 0.03 in males and 0.05 in females. Fifty-seven families gave positive lod scores; five small families gave mildly negative lod scores. The maximum likelihood estimate of alpha, the proportion of linked loci, was 1.0 with a lower 99% confidence interval of 0.88. These data suggest that there is only one HD locus, although a second rare locus cannot be ruled out.     

Identification and expansion of pancreatic stem/progenitor cells

Pancreatic islet transplantation represents an attractive approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells /progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstances, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even if the pancreas harbors a stem cell population need to be resolved. In this article, we review the recent achievements both in the identification as well as in the expansion of pancreatic stem/progenitor cells.     

Amplification methods for the immunolocalization of rare molecules in cells and tissues

The needs to precisely assign macromolecules to specific locations and domains within tissues and cells and to reveal antigens which are present in low or even in trace amounts, led to the elaboration of a wide spectrum of immunocytochemical amplification procedures. These arise from the successive improvements of tissue preparation techniques, of antigen retrieval procedures and of immunological or non-immunological detection systems. Improvement of detection systems may be the most active in the development of amplification techniques. Since the early work of Coons, in which by the introduction of the indirect technique has started amplifying the signal, different systems have succeeded in increasing the sensitivity of antigens detection. Indeed, amplification techniques such as the multiple antibody layers, the multiple bridges, the enzyme complexes, the avidin-biotin, the silver intensification, and the numerous variations and combinations among these have increased the sensitivity for the detection of scarce tissue antigens. However, as shown by the recent progress carried out with new approaches such as the catalyzed reporter deposition (CARD) and the enhanced polymer one-step staining (EPOS), more efficient methods are still needed. In electron microscopy, few techniques have reached the resolution afforded by the post-embedding immunogold approach. In spite of this and in order to further increase its sensitivity, new probes and novel approaches are allowing combination of the gold marker with the amplification capacity of enzymes afforded by the CARD technique. Immunogold amplification strategies, such as the multiple incubations with the primary antibody and the use of an anti-protein A antibody have also led to enhanced signals displaying the advantages in terms of resolution and possibilities of quantification inherent to the colloidal gold marker.     

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 μM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT).     

Cytokinin content and immunolocalization in wheat primary root cells

Immunohystochemical localization of cytokinins in cells of different root zones of wheat plants showed intensive immunostaining of zeatin in the apical root zone and its subsequent decline with the increase in the distance from the root tip. More intensive labeling of metaxylem and parenchyma cells of the root central cylinder was observed on the sections of the zone where root hairs appeared. Above this zone the decline in immunostaining of the cells of the central cylinder was paralleled by the signs if finalization of differentiation of the xylem vessels shown by lignin deposition. The data of immunohystochemical staining were confirmed by the results of enzyme immunoassay of different cytokinin forms. Likely sources of zeatin accumulation are considered. Possibility of additional (alongside with that in apical root zone) synthesis of cytokinins in the vascular tissues of root and the role of cytokinins in stimulation oflignification are discussed.     

Sympatric speciation in Yponomeuta: no evidence for host plant fidelity

According to sympatric speciation theory, adaptation to different host plants is expected to pleiotropically lead to assortative mating, an important factor in the reduction of gene flow between the diverging subpopulations. This scenario predicts mating on and oviposition preference for the respective hosts in both the diverging subpopulations and recently originated species. Here, we test both predictions in the oligophagous Yponomeuta padellus (L.) and the monophagous Yponomeuta cagnagellus (Hübner) (Lepidoptera: Yponomeutidae), two closely related small ermine moth species from the western European clade of Yponomeuta for which speciation in sympatry has been proposed. Mating location and adult host acceptance were evaluated under both semi-field (in a large outdoor cage with a choice of host and non-host plants) and field conditions. In the semi-field experiment, only Y. cagnagellus showed some preference for mating on its own host (16% of all mating pairs) over non-host plants (3% of all mating pairs). However, in both species, more than 80% of the mating pairs were not formed on a plant but instead on the cage itself. Further examination of the mating site of Y. cagnagellus in the field revealed no preference for host plants over non-host plants in the two consecutive years of observation. Yponomeuta padellus females, collected from and reared on Prunus spinosa L. (Rosaceae), showed an oviposition preference for the alternative host Crataegus monogyna Jacq. (Rosaceae) in the semi-field experiment. We thus found no evidence that host-plant fidelity (in terms of mating site) has been the driving force in the speciation process of these Yponomeuta species, nor did we find evidence of host race formation in the tested population of the oligophagous Y. padellus .     

Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen

Gametophytic tissues of plants are an area largely neglected in the broad literature on free radical processes in plants. In order to study the mechanisms of protection against oxidative stress in pollen, the presence of the key antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) was investigated. Crude extracts of olive tree ( Olea europaea L.) pollen were subjected to native PAGE in 10% polyacrylamide gels. The SOD activity staining of gels showed the presence of four isoenzymes. All the SODS were completely inhibited by 2 m M KCN and 5 m M H₂O₂, and therefore belong to the family of CuZn‐SODS. Isoelectric focusing (pH 3.5‐7) of crude extracts and further detection of SOD activity allowed determination of isoelectric points for the four isoforms, namely 4.60, 4.78, 5.08 and 5.22. The cross‐reactivity of pollen extracts with a polyclonal antibody to cytosolic CuZn‐SOD from spinach leaves was assayed by western blotting. After SDS‐PAGE and immunoblotting, a major polypeptide band of about 16.5 kDa was detected, which is characteristic of the subunit of most CuZn‐SODS. Immunocytochemical studies at TEM level using the same antiserum showed that CuZn‐SOD was localized in the cytoplasm of both vegetative and generative cells, and also in material adhered to the pollen wall. The olive pollen CuZn‐SODS could function in the protection against oxidative stress during pollen development.     

Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival

Calreticulin is a lectin chaperone essential for intracellular calcium homeostasis. Deletion of calreticulin gene compromises the overall quality control within the endoplasmic reticulum (ER) leading to activation of the unfolded protein response. However, the ER structure of calreticulin deficient cells (crt-/-) is not altered due to accumulation of misfolded proteins. Therefore, the aim of this study was to determine whether the ubiquitin-proteasome pathway is activated in crt-/- cells as a compensatory mechanism for cell survival. Here we show a significant increase in the expression of genes involved in ER associated degradation and activation of the ubiquitin-proteasome system in crt-/- cells. We also demonstrated that the ubiquitination of two proteins processed in ER, connexin 43 and A1AT NHK (alpha1-antitrypsin mutant) are increased in crt-/- cells. Furthermore, we showed that the increased proteasome activity in the crt-/- cells could be rescued upon re-introduction of calreticulin or calsequestrin (a muscle calcium binding protein). We also illustrated that increased cytosolic Ca2+ enhances the proteasome activity. Interestingly, suppression of calnexin function using siRNA further elevated the proteasome activity in crt-/- cells. This is the first report to show that loss of calreticulin function enhances the ubiquitin-proteasome activity which could function as a compensatory mechanism for cell survival.