A new morphological aspect of the brush cells of the mouse gallbladder epithelium

Summary The brush cells of the gallbladder epithelium of the mouse have microvilli not only at their luminal border but also on their lateral surface, from the level of the nucleus to the junctional complex. The lateral microvilli radiate from the brush cell in all directions, contain a core of filaments, and penetrate up to 3 m into the adjacent cells. The microvilli in these locations display small desmosomes at their base.     

Protein kinase C activity in mouse eggs regulates gamete membrane interaction

Gamete membrane interaction is critical to initiate the development of a new organism. The signaling pathways governing this event, however, are poorly understood. In this report, we provide the first evidence that protein kinase C activity in mouse eggs plays a crucial role in the regulation of this process. Stimulating PKC activity in mouse eggs by phorbol 12-myristate 13-acetate (PMA) drastically inhibited the egg's membrane ability to bind and fuse with sperm. Surprisingly, this significant reduction of gamete membrane interaction was also observed in eggs treated with the PKC inhibitors staurosporine and calphostin c. In further analysis, we found that while no change of egg actin cytoskeleton was detected after either PMA or calphostin c treatment, the structural morphology of egg surface microvilli was severely altered in the PMA-treated eggs, but not in the calphostin c-treated eggs. Moreover, sperm, which bound but did not fuse with the eggs treated with the anti-CD9 antibody KMC8, were liberated from the egg membrane after PMA, but not calphostin c, treatment. Taken together, these results suggest that egg PKC may be precisely balanced to regulate gamete membrane interaction in a biphasic mode, and this biphasic regulation is executed through two different mechanisms.     

Prominin‐1 (CD133) modulates the architecture and dynamics of microvilli

Prominin‐1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non‐epithelial cells. Irrespective of the cell type, prominin‐1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin‐Darby canine kidney cells as the main model. Our high‐resolution analysis revealed that upon the overexpression of prominin‐1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob‐like morphologies were observed and stimulated by mutations in the ganglioside‐binding site of prominin‐1. The altered phenotypes were caused by the interaction of prominin‐1 with phosphoinositide 3‐kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin‐1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin‐1‐ganglioside membrane complexes, phosphoinositide 3‐kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin‐1 and its mutants modified the structure of filopodia emerging from fibroblast‐like cells and silencing human prominin‐1 in primary hematopoietic stem cells resulted in the loss of uropod‐associated microvilli. Altogether, these findings strengthen the role of prominin‐1 as an organizer of cellular protrusions.      

Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells

Hybridoma cell lines were prepared from spleen cells of mouse immunized with mitotic HeLa cells. A monoclonal antibody (mH1), which intensively reacted with cleavage furrows of dividing HeLa cells in immunofluorescence, was obtained. In interphase, this antibody diffusely stained whole HeLa cells. Immunoelectron microscopy showed that mH1 antigens were localized at microvillus projections at the surface of dividing HeLa cells, but definite localization of that antigen was not observed in interphasic cells. Immunoblot analysis showed that mH1 is reactive to 42-kDa and 130-kDa components. Further, the 42-kDa component was identified as a gamma-actin homolog by N-terminal amino acid sequence analysis.     

Integument of the tapeworm scolex. 1. Freeze-fracture of the syncytial layer, microvilli and discoid bodies

The tegument of cestodes is the most important and structurally complex metabolic interface between these parasites and the hostile environment in which they reside. In spite of the complex metabolic, regulatory and immunological properties of this layer of syncytial cytoplasm, which are relatively well known, the detailed fine structural anatomy of the cestode tegument remains equivocal. The present study therefore reports the freeze-fracture morphology of the tapeworm (Hymenolepis diminuta) tegument. The most important features revealed by analysis of platinum replicas of freeze-fractured tapeworm scolex-neck tegument include: (a) presence of highly ordered linear and/or circumferentially-orientated rows of intramembrane particles situated on the PF fracture face of microvillar plasma membrane, which may participate in movements of the microvilli, (b) presence of apparent 'pores' (11 nm in diameter) at the tips of the tegumentary microvilli, which could serve as regulated gates through which extramicrovillar surface coating materials can be extruded, and (c) the alignment of cytoplasmic discoid bodies into positions at the bases of the surface microvilli such that they could move into the core of each microvillus and thereby release their contents for extrusion (via the pores) onto the outer surface of the microvilli. Concomitantly, the limiting membrane of the discoid bodies could be added to the tegument plasma membrane and thereby contribute to the rapid turnover of the tegumentary surface. This study provides the first detailed account of the ultrastructural anatomy of the tapeworm tegument and is intended to serve as a point of reference for future investigations of tapeworm tegumentary functions.     

Parapodial glandular organs in Spiophanes (Polychaeta: Spionidae)—studies on their functional anatomy and ultrastructure

Parapodial glandular organs (PGOs) of Spiophanes (Polychaeta: Spionidae) were studied using light and electron microscopy. These organs are found in parapodia of the mid body region, starting on chaetiger 5 and terminating with the appearance of neuropodial hooks (chaetiger 14 or 15 in adult individuals). Large PGOs in anterior chaetigers display different species‐specific types of openings whereas small PGOs in posterior parapodia of the mid body region always open in a simple vertical slit. Each PGO is composed of three main complexes: (1) the glandular sac with several distinct epithelia of secretory cells and secretory cell complexes and the reservoir filled with fibrous material, (2) the gland‐associated chaetal complex (including the region of chaetoblasts and follicle cells, follicular canals, two chaetal collector canals, the combined conducting canal, the chaetal spreader including the opening of the glandular organ with associated type‐1 secretory cells, and the gland‐associated chaetae), and (3) a bilayered musculature surrounding the gland. A considerable number of different cell types are involved in the secretory activity, in the guidance of the gland‐associated chaetae, and in the final expulsion of the fibrous secretion at the opening slit. Among these different cell types the type‐5 secretory cells of the proximal glandular complex with their cup‐shaped microvilli emanating thick microfibrils into the lumen of the glandular sac are most conspicuous. Secretory cells with cup‐shaped microvilli being involved in the production of β‐chitin microfibrils have so far only been reported from some representatives of the deep‐sea inhabiting Siboglinidae (Polychaeta). We suggest that the gland‐associated chaetae emerging from inside the PGOs of Spiophanes are typical annelid chaetae formed by chaetoblasts and follicle cells. Functional morphology implies the crucial role of PGOs in tube construction. Furthermore, the PGOs are discussed in consideration of phylogenetic aspects. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Surface features of Entamoeba histolytica and rabbit kidney (RK13) cell surface changes after trophozoite contact--observations by scanning electron microscopy.

Trophozoites of Entamoeba histolytica grown on glass, fixed in situ, and examined by scanning electron microscopy (SEM) were seen to possess microfilopodia on their lower sides and under surfaces, a hitherto undescribed feature. No surface lysosomes were seen. After trophozoites had been brought into contact with a host monolayer of cultured RK13 cells, immediate surface changes were observed. First the RK13 cell surface microvilli elongated, then decreased in number and finally disappeared. These changes were accompanied by erosion of the cell surface and cellular swelling.     

Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins.

Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.     

Differentiated properties characteristic of renal proximal epithelium in a cell line derived from a normal monkey kidney (JTC-12)

Summary The JTC-12 cell, an established cell line derived from a normal monkey kidney, was studied in an attempt to characterize the epithelial qualities. Phase contrast microscopy showed dome formation in confluent monolayers and electron microscopic examinations revealed the presence of numerous microvilli on the apical membranes and desmosome between cells. Sonicated cells showed activities of γ-glutamyl transpeptidase, leucine aminopeptidase, alkaline phosphatase, and trehalase, marker enzymes of renal proximal epithelium. Alkaline phosphatase activity exhibited the characteristics of a renal type isozyme. Furthermore, confluent JTC-12 monolayers exhibited Na⁺-dependent transport of hexose, amino acid as well as inorganic phosphate. These findings indicate that JTC-12 cells in monolayer culture maintain ultrastructural, biochemical, and physiological properties of renal proximal epithelial cells. This cell line will be useful for further studies on cellular functions of renal proximal epithelium. This work was supported in part by grants from The Ministry of Health and Welfare of Japan and from the Ministry of Education, Science and Culture of Japan.     

Isolation of a calcium-sensitive, 35,000-dalton microfilament- and liposome-binding protein from ascites tumor cell microvilli: identification as monomeric calpactin

Microvilli isolated from the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium-sensitive microfilament-binding protein, AMV-p35 (ascites microvillar p35). Association of AMV-p35 with microfilament cores during Triton X-100 extraction of the microvilli is half-maximal at 0.1-0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion-exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion-exchange column. AMV-p35 binds to phosphatidylserine liposomes and F-actin with half-maximal calcium concentrations of about 10 microM and 0.2 mM, respectively. Treatment of AMV-p35 with chymotrypsin yields a 33,000-dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV-p35 with anti-calpactin I and anti-lipocortin II, but little reactivity toward anti-lipocortin I. The close relationship between AMV-p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV-p35. By gel filtration and velocity sedimentation analyses purified AMV-p35 is a 35,000-dalton monomer. Moreover, AMV-p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000-dalton menomer. These findings indicate that AMV-p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV-p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.