Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Oligolysine-based oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin

Dendritic cells are potent antigen-presenting cells that express several membrane lectins, including the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). To identify highly specific ligands for these dendritic cell receptors, oligosaccharides were converted into glycosynthons (Os1) and were used to prepare oligolysine-based glycoclusters, Os-[Lys(Os)]n-Ala-Cys-NH2. Clusters containing two to six dimannosides as well as clusters containing four or five pentasaccharides (Lewisa or Lewisx) or hexasaccharides (Lewisb) were synthesized. The thiol group of the appended cysteine residue allows easy tagging by a fluorescent probe or convenient substitution with an antigen. Surface plasmon resonance was used to determine the affinity of the different glycoclusters for purified mannose receptor and DC-SIGN, whereas flow cytometry and confocal microscopy analysis allowed assessment of cell uptake of fluoresceinyl-labeled glycoclusters. Dimannoside clusters are recognized by the mannose receptor with an affinity constant close to 106 liter.mol-1 but have a very low affinity for DC-SIGN (less than 104 liter x mol-1). Conversely, Lewis clusters have a higher affinity toward DC-SIGN than toward the mannose receptor. Dimannoside clusters are efficiently taken up by human dendritic cells as well as by rat fibroblasts expressing the mannose receptor but not by HeLa cells or rat fibroblasts expressing DC-SIGN; DC-SIGN-expressing cells take up Lewis clusters. The results suggest that ligands containing dimannoside clusters can be used specifically to target the mannose receptor, whereas ligands containing Lewis clusters will be targeted to DC-SIGN.     

“SIF” cells in the sympathetic ganglia of the bullfrog,Rana catesbeiana: variety in population and innervation

Summary Small intensely fluorescent (SIF) cells appeared singly or, more frequently, in variably-sized clusters in the sacroccygeal 8th and 9th sympathetic ganglia of the bullfrog. Smaller clusters containing only two to nine SIF cells accounted for 61% of 1773 clusters examined. The largest cluster contained 283 cells. The number of cells in individual ganglia also varied from 21 to 3332. SIF cells, solitary as well as in smaller clusters, received no distinct form of the synaptic contact. In contrast, the cells in larger clusters were frequently innervated by nerve endings that were similar in vesicular constitution to the nerve endings on principal ganglion (PG) cells. No synaptic contact was found between SIF cells and PG cells. SIF cells were also characterized by their location in the vicinity of blood capillaries with a continuous endothelium. p]Our observation seems to suggest that larger clusters of SIF cells receiving nerve endings are linked to a paracrine and/or endocrine system. Chemical influence via the blood stream and intraganglionic milieu for non-innervated SIF cells in the solitary or smaller clusters is a subject for speculation. An interneuronal role of SIF cells to relay stimuli to PG cells seems unlikely. The possible functions here assigned to SIF cells could be variable in efficiency depending on their population and density.     

Cytology of bronchial gland carcinoma

The cytologic findings in two adenoid cystic and three mucoepidermoid carcinomas of the bronchial tree are reported. In one case of adenoid cystic carcinoma, the diagnosis was made on a fragment of tumor tissue exfoliated in the patient's sputum. In the other case, brushing and aspirated materials yielded large clusters of small cells arranged around cystlike spaces containing globular basophilic mucuslike material. Fine needle aspirates from the two low-grade mucoepidermoid carcinomas showed clusters of malignant squamous cells containing mucus-secreting cells. The high-grade mucoepidermoid cancer yielded malignant squamous and glandular cells in aspirates.     

Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters

Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.     

Mammosomatotrophs develop within mammotroph clusters in bovine adenohypophysis

The existence and distribution of mammosomatotrophs (MS cells) containing growth hormone (GH) and prolactin (PRL) in bovine adenohypophysis were detailed by a combined method of mirror sections and immunohistochemical staining. MS cells always occurred in bovine adenohypophysis but their number was low. In the midsagittal plane, the cells were observed in the hind dorsal, hind ventral and fore ventral region abundant in GH and PRL cells. Whereas, in the zona tuberalis where GH and PRL cells were less frequent, MS cells were not detected. MS cells were invariably solitarily distributed within mammotroph (PRL cell) clusters but not within somatotroph (GH cell) clusters. The proportion of MS cells declined as the ages proceeded and the appearance was spatially related to the arrangement of PRL cells. These findings indicated that, in bovine adenohypophysis, MS cells were differentially distributed and occurred in PRL cell clusters. The results strongly suggest that MS cells originate in GH cells pre-existed within PRL cell clusters with special reference to the functional activation of PRL cells.     

Fetal and early post-natal development of the human spleen: from primordial arterial B cell lobules to a non-segmented organ

Immunohistological analysis of 31 human spleens from the 11th week of gestation to the early postnatal period suggested that fetal organ development may be preliminarily divided into four stages. At stage 0 the organ anlage contained erythrocyte precursors, few macrophages and almost no lymphocytes. Fetal spleens of stage I exhibited arterial vascular lobules and lymphocytes just began colonizing the organ. At stage II, B and T lymphocytes formed periarteriolar clusters. B cell clusters predominated, because B cells aggregated around the more peripheral branches of splenic arterioles, while T cells occupied the more centrally located parts of the vessels. The vascular lobules of stage I and II consisted of central arterioles surrounded by B cells, capillaries and peripheral venules. The lobular architecture slowly dissolved at late stage II when sinuses grew out from the peripheral venules into the centre of the lobule. Interestingly, the B cell accumulations around peripheral arterioles did not represent the precursors of follicles, but apparently persisted as periarteriolar B cell clusters in the adult splenic red pulp, while follicles containing FDCs developed at late stage II from B cells in direct contact to T cell clusters around larger arterial vessels. At stage III before birth the lobular architecture was no longer recognized. The chemokine CXCL13 was already present in vascular smooth muscle and adjacent stromal cells at stage I before B cells immigrated. CCL21, on the contrary, was only demonstrated in fibroblast-like cells supporting T cell clusters from stage II onwards.     

Cell interactions influence the fate of mouse blastomeres undergoing the transition from the 16- to the 32-cell stage

Newly formed polar and apolar 1/16 blastomeres were isolated and cultured singly, or in various combinations, through division to form 32-cell blastomeres. The morphology of the resulting cell cluster appeared to depend upon the nature and composition of the cell combination used. In most polar + apolar couplets, the polar cell enveloped the apolar cell, and following division, a 4/32 cluster was thereby generated containing two trophectoderm-like external cells derived from the polar cell and two ICM-like internal cells derived from the apolar cells. A polar cell cultured in isolation divided to give either two trophectoderm-like external cells or a trophectoderm-like cell and an ICM-like cell. Two polar cells cultured together generated clusters in which the ratio of trophectoderm-like:ICM-like cells was 4:0 or 3:1. Most apolar cells cultured together in couplets polarized, and generated 4/32 clusters containing either purely trophectoderm-like or a mixture of trophectoderm- and ICM-like cells. The results are consistent with the notion that continuing interactions between polar and apolar cells are necessary to maintain their respective fates as trophectoderm and ICM, and that in the absence of these interactions polar cells can generate ICM cells by a differentiative division and apolar cells can generate trophectoderm cells by polarizing in response to asymmetric cell contacts.     

Non-lipid osmiophilic granules in the presynaptic neuroplasma of the boutons

Summary Brain, spinal cord and peripheral (sensory and sympathetic) ganglia of cats and rats have been fixed in Susa, imbedded and impregnated on slides with a mixture of osmium tetroxide and egg albumen solution.This method produced small (about one micron) black granules in the boutons around the multipolar nerve cells in the cord and in the medulla. The granules were absent around the other nerve cells in the cortex (pyramidal cells, etc.) and in the peripheral ganglia.These osmiophilic granules may be clusters of synaptic vesicles containing transmitter substance. If the clusters are large enough, as in the boutons, they are visible under the light microscope, if they are smaller;, as in the smaller synaptic knobs, they remain invisible.If the osmiophilic granules are clusters of vesicles containing the transmitter substance, this substance might be an acidic amino acid.     

Adipose conversion of Ob17 preadipocytes : Relationships between cell division and fat cell cluster formation

Adipose conversion of Ob17 preadipocyte cells proceeds after confluence with the formation of fat cell clusters, due to the coexistence of cells susceptible or not to adipose conversion. In order to determine whether commitment to differentiation occurs after quiescence or during exponential growth, the spatial arrangement of Obl7 cells was destroyed at different times before and after confluence by trypsinization followed by cell reinoculation. The resulting distribution of lipid-filled cells, as compared to non-replated control cells, indicates that both insusceptible and susceptible cells are present during the growth phase in the absence of insulin. It is shown that the formation of fat cell clusters of large size is due to mitoses of susceptible cells during a limited period of time after confluence. Blockade of post-confluent mitoses by selective elimination of cells in the S phase abolishes the formation of clusters of large size, but single differentiated cells and clusters containing a few cells remain present. Therefore post-confluent mitoses are not necessary for the differentiation to occur, but rather they serve to amplify the proportion of adipose cells relative to non-adipose cells.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding

Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Lifetime-based F?rster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (<10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 +/- 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.     

Germ cell cluster formation and ovariole structure in viviparous and oviparous generations of the aphid Stomaphis quercus

The developing ovaries of S. quercus contain a limited number of oogonial cells which undergo a series of incomplete mitotic divisions resulting in the formation of clusters of cystocytes. Ovaries of viviparous generations contain 6 to 9 clusters, containing 32 cystocytes each, whereas ovaries of oviparous generations contain 5 clusters containing 45-60 cystocytes. During further development, clusters become surrounded by a single layer of follicular cells, and within each cluster the cystocytes differentiate into oocytes and trophocytes (nurse cells). Concurrently, cysts transform into ovarioles. The anterior part of the ovariole containing the trophocytes becomes the tropharium, whereas its posterior part containing oocytes transforms into the vitellarium. The vitellaria of viviparous females are composed of one or two oocytes, which develop until previtellogenesis. The nuclei of previtellogenic oocytes enter cycles of mitotic divisions which lead to the formation of the embryo. Ovarioles of oviparous females contain a single oocyte which develops through three stages: previtellogenesis, vitellogenesis and choriogenesis. The ovaries are accompanied by large cells termed bacteriocytes which harbor endosymbiotic microorganisms.     

Establishment and characteristics of a cell suspension culture from a moss,Atrichum undulatum

A green cell suspension culture was established from callus tissue obtained from a moss,Atrichum undulatum. This suspension culture was subcultured every ten days in Murashige and Skoog's liquid medium (MS) containing 10^?6 M 2, 4-D and 4% glucose. The suspended cells grew vigorously with the passage of subculture, maintaining small clusters made up of a few cells. In this suspension culture, round spore-like cells released from the cell clusters were found. The yield of protoplasts from the suspension culture was significantly higher than that of the conventional method using moss protonemata developed directly from spores.     

Hierarchy of lesion processing governs the repair,double-strand break formation and mutability of three-lesion clustered DNA damage

Ionising radiation induces clustered DNA damage sites which pose a severe challenge to the cell’s repair machinery, particularly base excision repair. To date, most studies have focussed on two-lesion clusters. We have designed synthetic oligonucleotides to give a variety of three-lesion clusters containing abasic sites and 8-oxo-7, 8-dihydroguanine to investigate if the hierarchy of lesion processing dictates whether the cluster is cytotoxic or mutagenic. Clusters containing two tandem 8-oxoG lesions opposing an AP site showed retardation of repair of the AP site with nuclear extract and an elevated mutation frequency after transformation into wild-type or mutY Escherichia coli. Clusters containing bistranded AP sites with a vicinal 8-oxoG form DSBs with nuclear extract, as confirmed in vivo by transformation into wild-type E. coli. Using ung1 E. coli, we propose that DSBs arise via lesion processing rather than stalled replication in cycling cells. This study provides evidence that it is not only the prompt formation of DSBs that has implications on cell survival but also the conversion of non-DSB clusters into DSBs during processing and attempted repair. The inaccurate repair of such clusters has biological significance due to the ultimate risk of tumourigenesis or as potential cytotoxic lesions in tumour cells.     

Chromosomes in Bacillus subtilis Spores and Their Segregation During Germination

Spores of a thymine-requiring mutant of Bacillus subtilis 168 leucine(-), indole(-), thymine(-)) were uniformly labeled with (3)H-thymidine. These were seeded on thinlayer agar plates where they germinated into long-chained microcolonies. Autoradiograms were used to measure the distribution of labeled deoxyribonucleic acid in the chains of cells, which ranged in length from 2 to 32 cells. Four major grain clusters appeared in most chains. These clusters were homogeneous in size; their grain numbers were distributed symmetrically from 9 to 15 with an average of 12.0. When three or fewer major clusters appeared in short chains, some of them were composed of two subclusters. However, there were always four clusters per chain when these subclusters were counted as individuals. Groupings containing two to eight grains appeared, as well as the four major clusters in longer chains. These minor groups were fragments of the major clusters. In contrast to the symmetrical distribution of major clusters, fragmented clusters were distributed at random, indicating random fragmentation. The total number of major and minor clusters increased at a constant exponential rate when measured against total cell number per chain, i.e., number of generations. It was calculated from the rate that a detectable fragmentation, at least 16% of a conserved unit (defined as a single strand of the complete chromosome), occurred every 6.0 generations. These results led us to conclude that each B. subtilis spore contained four conserved units or two completed chromosomes. Segregation of the four units into progeny cells was almost random. The one notable exception was a conserved unit which frequently appeared in a terminal cell to which an empty spore coat was attached. The presence of two chromosomes in the spore is consistent with our proposed structure of the completed chromosome, in which two sister chromosomes are covalently linked at the initiation region. This double chromosome may be incorporated into the spore without further structural change.     

c-Cbl-mediated regulation of LAT-nucleated signaling complexes

The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.     

Distinct Cell Clusters Touching Islet Cells Induce Islet Cell Replication in Association with Over-Expression of Regenerating Gene (REG) Protein in Fulminant Type 1 Diabetes

Background

Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear.

Procedures

The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans.

Result

Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells.

Conclusion

The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.     

Acetylcholine receptor clusters are associated with nuclei in rat myotubes

Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with alpha-bungarotoxin conjugated to tetramethylrhodamine (alpha BTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development.