Showing papers on "Apical cytoplasm published in 2015"

The Drosophila centriole - conversion of doublets into triplets within the stem cell niche

Abstract: We report, here, that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed by microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. We demonstrated that the old mother centriole, which stays in the apical cytoplasm of the male germline stem cells (GSCs), is invariably composed of triplets, whereas its daughter is always built of mixed doublets and triplets. This difference represents the first documentation of a structural asymmetry between mother and daughter centrioles in Drosophila GSCs and might reflect a correlation between the architecture of parent centrioles and their ability to recruit centrosomal proteins. We also found that the old mother centriole is linked to the cell membrane by distinct projections that might play an important role in keeping its apical position during centrosome separation.

The profile of the epidermal growth factor system in rat endometrium during postpartum involution period

Abstract: The epidermal growth factor (EGF) plays a crucial role in the control of uterine cell proliferation, growth and differentiation. This study was designed to investigate the spatiotemporal expression pattern and localization of the EGF receptor/ligand system during the process of uterine involution using immunohistochemistry. Our results indicated that the expression of the ErbB/HER receptors and their ligands varied with structural changes in the uterus at different days of involution. Supranuclear punctate ErbB1 immunostaining was observed in the luminal and glandular epithelial cells and endometrial fibroblasts. Moderate ErbB2/HER2 immunoreactivity was observed in the lateral membrane and cytoplasm of the epithelial cells on the 1st, 3rd and 5th days and was decreased on the other days of involution. The amount of nuclear and cytoplasmic ErbB3/HER3 and ErbB4/HER4 immunostaining remained constant throughout the postpartum period. The EGF immunoreaction was weak in the luminal and glandular epithelium throughout the involution period. Although the cytoplasmic AREG immunoreactivity in the glandular epithelium was stronger on the 1st and 3rd days compared with the other days of involution, NRG1 immunostaining was weak on the 1st and 3rd days and was moderate in the apical cytoplasm on the 10th and 15th days of involution. The macrophages displayed strong cytoplasmic immunoreactivity for ErbB3/HER3, ErbB4/HER4, EGF, AREG and NRG. Strong, moderate and weak immunostaining for ErbB2/HER2, ErbB4/HER4 and other proteins (ErbB1, ErbB3, AREG and NRG), respectively, was present in the myometrial smooth muscle cells. These findings support the hypothesis that the EGFsystem plays a role in the development of various physiological changes associated with uterine involution.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

GP2-expressing cells in the conjunctiva and tear ducts of mice: identification of a novel type of cells in the squamous stratified epithelium

Abstract: GP2 is a membrane-associated secretory protein originally identified in zymogen granules of pancreatic acinar cells. Recently, this glycoprotein has attracted attention as a marker substance of M cells of Peyer's patches and for its involvement in the selective uptake of pathological bacteria via M cells. When we stained the conjunctiva and tear ducts of mice using a GP2 antibody, all goblet cells in the squamous stratified epithelium of the conjunctiva were intensely immunolabeled, while goblet cells in the intestine and airway were devoid of the immunoreactivity, indicating that the conjunctiva contains a special type of goblet cell. Further immunostaining for GP-2 labeled dispersed cells of peculiar shapes within the stratified squamous epithelium in the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. The GP2-immunoreactive cells in the tear duct projected arched or branched processes toward the basement membrane. Electron-microscopically, immunogold particles for GP2 outlined the basolateral plasma membrane of both the conjuntival goblet cells and the peculiarly shaped cells in the tear duct. Intracellularly, GP2 products of the goblet cells were localized around secretory granules in the apical cytoplasm and those of the tear duct cells inside the vesicles. The luminal contents close to apical plasma membrane were heavily labeled with immunogold particles, suggesting an exocytosis-based targeting of GP2 to the plasma membrane and its release into the lumen. The possible function of GP2 in tear ducts is discussed in relation to a defense system against invasive microoranisms and antigens.

Basal body docking in airway ciliated cells.

Abstract: Multiciliated cells (MCCs) in the respiratory epithelium play crucial roles in the innate defense system against airborne pathogens and pollutants. Each MCC possesses 200-300 cilia on their apical surface, which beat in a synchronized manner to clear mucus with foreign particles from the respiratory tract. Ciliary dysfunction leads to serious respiratory diseases including chronic infection as seen in patients with primary ciliary dyskinesia (PCD), a ciliopathy with defective multicilia. However, the molecular mechanisms underlying the differentiation of MCCs are poorly understood. In MCC precursor cells, downregulation of Notch signaling induces expression of the coiled-coil protein Multicilin (Mcidas), which in turn upregulates expression of the ciliogenesis transcription factors Myb, RFX and FoxJ1 that direct MCC differentiation. The majority of centrioles are generated de novo via an acentriolar pathway that involves centriole nucleation from fibrogranular structures called deuterosomes. In contrast, a canonical centriolar pathway, in which centrioles replicate using the existing centriole as a template, contributes to a small portion of the total centriole population. Nascent centrioles mature by acquiring accessary structures, which include distal appendages (DAs). Upon docking to the apical membrane, centrioles and DAs transform into basal bodies and transition fibers (TFs), respectively. DAs/TFs are fibrous structures that emanate from the distal end of the B-tubules of each of the nine centriole/basal body triplet microtubules, and are important for membrane docking [1]. At the early stages of ciliogenesis, small vesicles termed DA vesicles (DAVs) are captured by DAs and then fuse with each other to form a large membranous cap, the so-called ciliary vesicle, which later fuses with the apical membrane to facilitate basal body docking [1, 2], although alternative pathways may also exist. The extension of cilia from the basal bodies is mediated by the intraflagellar transport (IFT) system. Chibby (Cby) is an evolutionarily conserved 15-kD coiled-coil protein that localizes to the base of cilia. We previously reported that Cby−/− mice suffer from chronic respiratory infection due to a lack of effective mucociliary transport [3]. Consistent with this phenotype, we further demonstrated that Cby−/− MCCs show a marked reduction in the number of cilia and consistently contain undocked basal bodies in the apical cytoplasm. Taken together, these data suggested that Cby is required for efficient basal body docking. However, the precise molecular function of Cby during MCC differentiation remained unknown. Recently, using SIM and STORM super-resolution microscopes, we found that Cby protein clusters at the base of mature cilia as a ring with a diameter of 300 nm and a height of 100 nm [4]. Intriguingly, immuno-EM revealed that Cby is enriched around the distal portion of TFs where the TFs contact the apical membrane. During early stages of MCC differentiation, Cby localizes to the DAs of migrating centrioles, which are the sites of DAVs recruitment. Notably, in Cby−/− MCCs, only small DAVs are bound to the DAs, which indicates that Cby is not essential for the DAV-DA attachment. Rather, Cby appears to be required for the subsequent fusion of DAVs into larger ciliary vesicles. Molecularly, we demonstrated that Cby is recruited to the DAs through a physical interaction with the DA protein CEP164. Cby then associates with the membrane trafficking component Rabin8, a guanine nucleotide exchange factor (GEF) for the small GTPase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Since the first morphological EM study on MCCs in rat lungs in the 1960s [2], the molecular basis for basal body docking has remained largely elusive. Our study revealed that Cby facilitates basal body docking by promoting proper formation of ciliary vesicles. Our findings also underscore the biological importance and requirement of ciliary vesicle formation for efficient basal body docking. We postulate that the ciliary vesicle system is particularly important for MCC differentiation and ciliogenesis as they must coordinate the docking of hundreds of basal bodies over a short period of time. Cby most likely mediates ciliary vesicle assembly during primary ciliogenesis in a similar manner as Cby depletion significantly reduces the number of primary cilia in various cell types [5, 6]. We are only beginning to understand the molecular details for ciliary vesicle assembly. Recently, the membrane shaping proteins EHD1 and EHD3 and the SNARE membrane fusion regulator SNAP29 have been shown to function in ciliary vesicle formation [7]. However, the relationship between Cby and these novel regulators is currently unclear. Further identification and characterization of DA components and their associated proteins are critical for broadening our understanding of ciliogenesis and the pathobiology of ciliopathies.

Ultrastructure of Developing Feline Nonciliated Bronchiolar Epithelial Cells.

Abstract: The ultrastructure of the developing bronchiolar cell was studied in six age groups: prenatal (60 d post-conception); postnatal (1-, 7-, 14- and 21-day-old); and adult. Following intratracheal fixation, the lung tissue was processed for scanning and transmission electron microscopy. The lining of terminal bronchioles consists of cuboidal to columnar nonciliated bronchiolar cells (NBCs) and ciliated with or without microvilli. NBCs were recognized by indented centrally located nucleus. The apical surface extended beyond the surface of neighboring cells and was covered by minute microvilli, except in prenatal kittens. The NBCs of the adult were characterized by abundant mitochondria and glycogen inclusions. In prenatal kittens, the cytoplasm was filled with patches of alpha and beta form of glycogen. Postnatally, glycogen was reduced in quantity, became scattered throughout the cytoplasm and was predominantly of the beta form. Islands of cytoplasm, separated from the apical cytoplasm were observed i...

Tissue organization and cell ultrastructure in the roots of three Arabidopsis species grown at different zinc concentrations

Abstract: The model plant Arabidopsis thaliana is known to be heavy metal-sensitive in contrast to its relative species A. arenosa and A. halleri classified as pseudometallophytes. Quantitative differences in primary root anatomy previously found between A. thaliana and the non-metallicolous (NM) and metallicolous (M) populations of the non-model Arabidopsis species necessitated further research at cellular and ultrastructural levels. Seedlings of A. thaliana, ecotype Columbia and a natural population Ratkovo, the NM and M populations of A. arenosa and A. halleri were grown on agar medium containing 10 µM (control) and 1000 µM Zn2+ for 5 days. Light microscopy confirmed the higher number of cells in the endodermal, cortical and epidermal layers and a higher incidence of additional cell tiers, the so-called middle cortex (MC) in the tolerant genotypes. Such differences were present in untreated plants and even more pronounced in plants exposed to excess of zinc (Zn). Electron microscopy of the root tissues at comparable distances from the root tip showed Casparian bands only in the radial cell walls of endodermis of A. halleri M population originating from severely (Cu, Cd and Pb) contaminated site. Casparian bands were not differentiated yet in the roots of the other species and populations, and they were not formed in the cell walls between endodermis and MC cells. In the apical cytoplasm of trichoblast bulges, autophagic vacuoles were found only in the sensitive A. thaliana and small vacuoles in the other genotypes. The enhanced concentration of Zn confirmed the higher metal sensitivity of the model species and did not substantially disturb the root cell ultrastructure of the tolerant Arabidopsis species.

Characterization of regulatory factors controlling tip growth in Nicotiana tabacum pollen tubes

Abstract: Rho GTPases constitute a family of highly conserved signaling proteins with key functions in the regulation of cellular polarization and polar cell growth. In plants, Rho GTPases are represented by a plant specific subfamily referred to as Rac/Rop GTPases. Some members of this subfamily associate with the plasma membrane specifically at the tip of pollen tubes and are important regulators of polar cell expansion. The aim of my study has been to improve our understanding of the molecular mechanisms that control the activity of pollen tube Rac/Rop, and the signaling downstream of this activity. BiFC (Bimolecular Fluorescence Complementation) is a technique used to validate protein-protein interactions. My studies on the interactions between Nt-RhoGAP1/ RhoGDI2 and Nt-Rac5 show that 35S promoter regulated BiFC works in normally growing N. tabacum pollen tubes and that the intracellular interaction sites between Nt-RhoGAP1/ RhoGDI2 and Nt-Rac5 are found at the subapical plasma membrane and apical cytoplasm, respectively. This technique was consequently used to confirm potential interacting protein partners involved in Nt-Rac5 signaling networks (i.e. Nt-Risap and Nt-Ric), and to detect the subcellular localization of these interactions in pollen tubes. Nt-Risap (Rac5 interacting sub-apical protein) and Ric (Rho interacting CRIB containing) were isolated as effectors of Nt-Rac5 in yeast two-hybrid screens. The two proteins were confirmed to be highly and specifically expressed in N. tabacum pollen tubes and to interact with Nt-Rac5 both in vitro and in vivo. Functional studies of the two proteins suggest that they behave as downstream effectors of Nt-Rac5 and are involved in the Nt-Rac5 signaling pathways that regulate pollen tube tip growth by activating specific downstream events.

Localization of Cannabinoid Receptor 1 (CB1) in Submandibular and Sublingual Salivary Glands of Mice throughout Postnatal Development Localización de los Receptores Cannabinoides 1 (CB1) en Glándulas Salivales Submandibulares y Sublinguales de Ratones Durante el Desarrollo Postnatal

Abstract: HIPKAEO, W.; WATANABE, M. & KONDO, H. Localization of cannabinoid receptor 1 (CB1) in submandibular and sublingualsalivary glands of mice throughout postnatal development. Int. J. Morphol., 33(2):695-700, 2015.SUMMARY: In view of the fact that human marijuana users often show dry mouth symptom, the present study was attemptedto examine the localization of CB1, which was originally identified in brain, in the submandibular and sublingual salivary glands ofpostnatal developing male mice by immunohistochemistry. In submandibular gland, CB1-immunoreactivity was positive in a majorityof acinar cells in forms of granular appearance in their apical cytoplasm, while it was negative in the ducts at newborn stage. Theimmunoreactivity decreased in the acinar cells at P1W and no immunoreactivity was detected in the acinar cells at P3W and there after.The immunoreactivity was positive in ductal cells at P3W and it remained positive thereafter until P8W stage. The immunoreactio n wasdistinct on the apical plasmalemma of the intercalated ductal cells, while it was distinct on the basal plasmalemma of the granularconvoluted ductal cells. The enhanced immunostaining on the lateral plasmalemma of the granular ductal cells was discerned only onP6W. In sublingual gland, CB1-immunoreactivity was detected in the demilune acinar cells and ductal cells only on P4W. Furtherm ore,CB1-immunoreactivity was shown to occur in the salivary ganglionic neurons, suggesting the CB1-inhibitory action in the saliva secretionthrough the parasympathetic nervous transmission.KEY WORDS: Cannabinoid receptor 1; Immunohistochemistry; Submandibular gland; Sublingual gland; Postnatal mice.

Histochemical characteristics of glycoproteins during rat palatine gland development

Abstract: Lectin histochemistry has been used to investigate glycosylation modification and glycoprotein expression that occurs during development and under different physiological and pathological conditions. Several lectin histochemical studies have been performed on the palatine gland of different species, which have described the heterogeneity of complex glycoconjugates present in these structures. However, no study has been conducted with regard to the relationship between glycoproteins and palatine gland development in mammals. Therefore, we conducted a study to test the hypothesis that a considerable modification in the expression of carbohydrates occurs in the palatine gland during developmental differentiation and maturation. Histochemical changes of glycoconjugates were observed during prenatal and postnatal development of the rat palatine gland. Qualitative and quantitative differences for the binding of lectins to palatal epithelium sections were determined. All lectins showed general progressive staining during development that was basally extended from the apical cytoplasm of mucous cells. The distribution of glycoproteins during palatine gland development and the heterogeneous distribution of glycoproteins observed between posterior and anterior sides expand our knowledge of the role of salivary glands in oral function. In this review, we describe and discuss glycohistochemical observations of the developing rat palatine gland.