A cytochemical study of GERL in cultured human fibroblasts

Cultured human skin fibroblasts contain an extensive GERL network. The GERL consists of acid phosphatase-positive polygonal tubules and tuberous thickenings which are adjacent to the Golgi cisternae. It often surrounds lucent vacuoles and rarely lipid bodies. Cultures examined at various intervals after subculturing, passages in culture and at different ages of the source of the primary fibroblasts demonstrate no significant morphologic variation in GERL. However, the network appears to fragment in mitotic fibroblasts. Cultured fibroblasts from patients with lysosomal storage diseases exhibit a normal GERL. Exposure of fibroblasts to non-toxic levels of chloroquine and ammonium chloride result in increasing dilatation of the GERL and the resultant formation of large cytoplasmic vacuoles. These observations support the previously proposed theory that this network is a special form of lysosome packaged to contain a large membrane pool.     

Ultrastructural study of GERL in beige mouse alveolar macrophages

Alveolar macrophages of the beige mouse mutant have a system of smooth- surfaced elements with the hallmarks of GERL. GERL also appears to produce residual bodies, and both organelles show cytochemically demonstrable acid phosphatase activity. When cells are exposed to colloidal silver, the tracer is endocytosed via pinocytic vacuoles to GERL.     

Bifunctional properties of lectins: lectins redefined

Both ‘classical’ lectins, originally identified as cell agglutinins, and other carbohydrate-binding proteins that were identified by different means, may contain a second type of binding site that is specific for a non-carbohydrate ligand. This, among other findings, is changing our view of endogenous lectin functions and of the proper definition of this group of proteins.     

The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.     

Cytochemical contributions to differentiating GERL from the Golgi apparatus

Synopsis Recent studies from our laboratory are described which deal with endocrine cells (insulinoma, -cells of the pancreas, thyroid epithelial cells), pancreatic exocrine cells, and hepatocytes. These emphasize the importance of the hydrolase-rich specialized region of endoplasmic reticulum, known as GERL, in secretory cells. Also reviewed in this paper are the varied molecular transformations which apparently occur in GERL in different cell types, as reported from other laboratories as well as our own. Evidence of the continuity of GERL with rough endoplasmic reticulum is presented. Two hydrolytic enzyme activities in GERL, in addition to acid phosphatase activity, are recorded. Finally, the use of cytochemical staining procedures in the study of microperoxisomes is briefly described. The Histochemical Journal lecture 1976. Delivered to the Histochemistry and Cytochemistry Section of the Royal Microscopical Society on 14 September 1976     

C型凝集素

C型凝集素（C-type lectin）代表一个识别碳水化合物配体依赖于钙离子（Ca2＋）参与的糖原结合蛋白家族,含有一个或多个一级结构和二级结构同源的碳水化合物识别结构域。随着研究的深入,越来越多的C型凝集素能够识别体内的非糖类的配体,包括蛋白质和脂类等。这些C型凝集素在维持机体稳态、免疫防御以及免疫监视等重要生理病理过程中发挥着重要作用。就C型凝集素的结构、分类和在免疫系统中的功能作一介绍。     

Bean lectins

Summary The relationship between the polypeptide composition and the agglutination behaviour of the lectin-containing G2/albumin protein groups has allowed the identification of the active lectin polypeptides in different cultivars of Phaseolus vulgaris (Brown et al. accompanying paper). These results were used to ascertain the particular G2/albumin group contained in the various lectin sources used previously for the purification of lectin proteins. Many studies were found to have included lectin sources which contained the same G2/albumin pattern (T_G2) and this common denominator has permitted the direct comparison of the properties reported for these purified lectins. Thus, much of the extensive literature on bean lectins is concurred.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Internalization of seasonal time

Orientation of physiology and behavior in time is a major adaptation common to many organisms and represents a significant challenge. In the face of predictable seasonal changes in climatic factors, and as a result of natural selection, many mammals now restrict their reproductive efforts to the fraction of the year when conditions of temperature, food, and water are most favorable for successful weaning of offspring. Changes in day length figure prominently in the synchronization of mammalian seasonal reproductive cycles. In summer breeders (e.g., several hamster, vole, and mouse species), decreasing summer day lengths induce reproductive involution. During this interval, neuroendocrine mechanisms analogous to a simple reference memory permit discrimination of stimulatory from inhibitory photoperiods. The non-reproductive phenotype is sustained for several months thereafter by the inhibitory short days of late summer, autumn, and early winter. Mid-winter reactivation of the reproductive system is triggered after 20-25 weeks of exposure to decreasing or short days by an interval timer that renders the reproductive neuroendocrine system refractory to short days. Recent work that has explored formal and physiological properties of this photorefractoriness interval timer has identified sex differences, neural substrates, and changes in hypothalamic gene expression that may participate in the measurement of seasonal time.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

I-type lectins

The immunoglobulin superfamily is a large category of proteins defined by their structural similarity to immunoglobulins. The majority of these proteins are involved in protein-protein binding as receptors, antibodies or cell adhesion molecules. The I-type lectins are a subset of the immunoglobulin superfamily that are capable of carbohydrate-protein interactions. There are I-type lectins recognizing sialic acids, other sugars and glycosaminoglycans. The occurrence, structure, binding properties and (potential) biological functions of the I-type lectins are reviewed here.     

Nucleocytoplasmic lectins

This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase alpha. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.     

Internalization of Echovirus 1 in Caveolae

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.     

Bean lectins

Summary Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F₂ plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.     

Bean lectins

Summary Single seeds of over 100 bean cultivars were analyzed by two-dimensional electrophoresis. The cultivars could be classified into eight groups by virtue of their G2/albumin electrophoretic patterns: T_G2, S_G2, V_G2, Pr_G2, B_G2, M_G2, P_G2, and Pi_G2, The polypeptide compositions of these types were largely inter-related having particular polypeptides in common. It was possible to correlate the G2/albumin patterns with agglutinating activity of cow and rabbit blood cells as measured by the agglutination ratio (minimum concentration of extract required to agglutinate cow blood cells: minimum concentration of extract required to agglutinate rabbit blood cells). The active lectin polypeptides were identified by extracting lectins from agglutinated erythrocytes and by comparing the qualitative similarities and differences of the G2/albumin patterns and their agglutination activities. A reference catalogue of over 100 bean cultivars giving their phaseolin and G2/albumin electrophoretic patterns, and agglutination ratios is presented.     

Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.