The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell differentiation

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane protein concentrated in plasma membrane protrusions. In epithelial cells, notably neuroepithelial stem cells, prominin-1 is found in microvilli, the primary cilium and the midbody. These three types of apical membrane protrusions are subject to remodeling during (neuro)epithelial cell differentiation. The protrusion-specific localization of prominin involves its association with a distinct cholesterol-based membrane microdomain. Moreover, the three prominin-1-containing plasma membrane protrusions are the origin of at least two major subpopulations of prominin-1-containing extracellular membrane particles. Intriguingly, the release of these particles has been implicated in (neuro)epithelial cell differentiation.     

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.     

Echinophilic proteins stomatin, sorcin, and synexin locate outside gangliosideM1 (GM1) patches in the erythrocyte membrane

The detergent (Triton X-100, 4 °C)-resistant membrane (DRM)-associated membrane proteins stomatin, sorcin, and synexin (anexin VII) exposed on the cytoplasmic side of membrane were investigated for their lateral distribution in relation to induced ganglioside_M1 (GM1) raft patches in flat (discocytic) and curved (echinocytic) human erythrocyte membrane. In discocytes, no accumulation of stomatin, sorcin, and synexin in cholera toxin subunit B (CTB) plus anti-CTB-induced GM1 patches was detected by fluorescence microscopy. In echinocytes, stomatin, sorcin, and synexin showed a similar curvature-dependent lateral distribution as GM1 patches by accumulating to spiculae induced by ionophore A23187 plus calcium. Stomatin was partly and synexin and sorcin were fully recruited to the spiculae. However, the DRM-associated proteins only partially co-localized with GM1 and were frequently distributed into different spiculae than GM1. The study indicates that stomatin, sorcin, and synexin are echinophilic membrane components that mainly locate outside GM1 rafts in the human erythrocyte membrane. Echinophilicity is suggested to contribute to the DRM association of a membrane component in general.     

Preparation and characterization of monoclonal antibodies recognizing three distinct differentiation antigens (BL1, BL2, BL3) on human B lymphocytes

We report in this paper the generation and characterization of three monoclonal antibodies, designated alpha BL1, alpha BL2, and alpha BL3, that recognize distinctive antigens unrelated to complement, Fc, and mouse erythrocyte rosette receptors, which are preferentially expressed on B lymphocytes. alpha BL1 recognizes a heat stable nonimmunoprecipitable antigen, possibly glycolipid in nature. Alpha BL2 recognizes a nonreducible single polypeptide with a m.w. of 68,000 that occasionally co-precipitates with a p29,34 complex of HLA-DR antigens. Alpha BL3 recognizes a nonreducible single polypeptide with a m.w. of 105,000 with an acidic pI point. We demonstrated that BL1 is expressed on fetal liver hematopoietic cells, a small subset (5 to 15%) of Ficoll-Hypaque-separated normal bone marrow cells, and on a subpopulation of nonadherent, non-E rosette-forming cells and granulocytes. BL2 is expressed on fetal liver hematopoietic cells, on 3 to 7% of normal bone marrow cells, and on a majority (40 to 70%) of nonadherent, non-E rosette-forming cells with a distinctive pattern similar to that of HLA-DR. BL3 is expressed on a subpopulation of nonadherent, non-E rosette-forming cells, and on occasional cells in the monocyte-enriched adherent cell population. The peak fluorescence for BL2 is substantially higher than that of BL1 and BL3, indicating higher BL2 antigen density. All three antigens are absent from thymocytes and E rosette-positive T cell fractions obtained from various lymphoid tissues. Cellular distribution of the BL antigens on various well-characterized established hematopoietic cell lines, leukemias, and malignant lymphomas, in conjunction with the results of the in vitro activation and TPA-induction experiments, suggest that BL1 is expressed during early developmental stages of B cell differentiation, whereas BL3 is expressed at the later stages. BL2 expression spans immature and mature stages of B cell differentiation, with the exception of mature plasma cells. The alpha BL antibodies described here should prove to be useful in the investigation of B cell differentiation and in the clinical diagnosis of lymphoid neoplasms.     

Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.