Microtubular system in cultured mouse epithelial cells

Previous observations had shown that colcemid does not affect locomotion of epithelial cells. Nevertheless, cultured mouse kidney cells forming epithelial sheets were found to contain a well-developed microtubular system sensitive to colcemid. The orientation of microtubules in the epithelial cells was not correlated with the stable or active state of the cell edges. It is suggested that microtubular system of the epithelial cells forming coherent sheets, in contrast to that of individually moving fibroblasts, is not essential for stabilization of the lateral cell edges.     

Morphology of the microtubule system during the sphering, layering and polarization of normal and transformed fibroblasts]

The ultrastructure of the genital system (ductus seminalis, vestibulum, ductus tortuosus and canaliculus fecundans) has been studied during mating and oviposition. All these organs have a common organization and contain three parts: muscular and epithelial layer and chitin intima lining the interior surface of the female genital system. Ultrastructures of the muscular tissue and of the basal part of epidermis are similar. The luminal surface and microvilli are different in various organs of the female genital system. The ultrastructural pattern and the thickness of chitin intima may also vary, and can be used as a character for identificating different organs of the female system in ultrathin sections.     

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells

Summary Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca²⁺ medium (1.05 mM). To clarify the mechanism by which Ca²⁺ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca²⁺ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca²⁺ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca²⁺ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca²⁺-grown cells did not display a network of tubulin fibers, whereas high Ca²⁺-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca²⁺ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatement of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca²⁺ medium had no observable effect on cell morphology. These results indicate that high levels of Ca²⁺ either favor tubulin polymerization or stabilize the polymerized state. This research was supported by NCI grant CA-38921 from the National Cancer Institute, Bethesda, MD, and by an institutional grant from the United Foundation of Greater Detroit.     

Effect of hydrocortisone on the mitotic cycle of epithelial cells in the mouse pylorus]

It was shown with the aid of thymidine-H3 that the mitotic cycle of mucous-forming cells (superficial epithelial mucosal cells of the neck) of the stomach pyloric glands of mice lasted 13.5 hrs (G1+1/2M = 7.6 hrs, S = 5.3 hrs; G2+1/2M = 0.6 hrs). With the administration of a physiological dose of hydrocortisone (0.1 mg) the duration of the mitotic cycle of mucous-forming cells of the stomach pyloric glands increased by 6.7 hrs (G1+1/2M = 11.6 hrs, S = 7.8 hrs; G2+1/2M = 0.8 hrs). A high dose of the hormone had a similar effect and increased the presynthetic period to 12.9 hours and the postsynthetic one--to 2.3 hours.     

Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment

Breast tumor cells enter the bloodstream long before the development of clinically evident metastasis. However, the early presence of such bloodborne cells predicts poor patient outcome. Nearly 90% of human breast tumors arise as carcinomas from mammary epithelial cells, so it is important to study how these cells respond to the detached conditions that they would experience in the bloodstream. We report here that mammary epithelial cell lines produce long and dynamic protrusions of the plasma membrane when detached. Although human and mouse mammary epithelial cell lines die by apoptosis within 16 h of detachment, this protrusive response persists for days in cells overexpressing either Bcl-2 or Bcl-xL. Unlike actin-dependent invadopodia and podosomes, these protrusions are actually enhanced by actin depolymerization with Cytochalasin-D or Latrunculin-A. Immunofluorescence and Western blotting demonstrate that the protrusions are enriched in detyrosinated Glu-tubulin, a post-translationally modified form of alpha-tubulin that is found in stabilized microtubules. Video microscopy indicates that these protrusions promote cell-cell attachment, and inhibiting microtubule-based protrusions correlates with reduced extracellular matrix attachment. Since bloodborne metastasis depends on both cell-cell and cell-matrix attachment, microtubule-based protrusions in detached mammary epithelial cells provide a novel mechanism that could influence the metastatic spread of breast tumors.     

The unusual microtubule polarity in teleost retinal pigment epithelial cells

In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule-dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types.     

Differentiation of epithelial cells in the mouse thymus

Summary The foetal and post-natal development of the mouse thymus was studied with the electron microscope paying particular attention to the differentiation of the epithelial cells. At about 13 days' gestation, the thymus was composed principally of undifferentiated epithelial cells and some lymphoblasts. The latter accumulated rapidly but did not show much evidence of mitotic activity until after the development of differentiated cortical epithelial cells which appeared during the 15th day of gestation. Further differentiation of epithelial cells did not occur until near term when medullary cystic epithelial cells appeared, and post-natally when small Hassall's corpuscles were developed. Undifferentiated and dividing epithelial cells were seen in the medulla and were present in all postnatal animals examined.This is publication number 1400 from the Walter & Eliza Hall Institute of Medical Research.The author is grateful to Prof. G. J. V. Nossal, Dr. J. F. A. P. Miller and Dr. P. J. Russell for their interest and assistance with various aspects of this study. Special thanks are due to Miss Mary Bravington for her skilled technical assistance. This investigation was supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and the National Health and Medical Research Council of Australia. The Electron Microscope Laboratory was equipped and supported by grants from the Australian Research Grants Committee, J. B. Were and Sons and the Potter Foundation.     

CAMSAP3-dependent microtubule dynamics regulates Golgi assembly in epithelial cells

The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.     

Immunochemical study of the CBA mouse kidney]

Immunoelectrophoresis and agar precipitation reaction allowed one to reveal 7 antigens in CBA mouse kidney. One of them, with a relative electrophoretic mobility--0.35, is specific for the kidney. The remaining renal antigens are common to the kidney and other organs and tissues of mice. The antigens with mobility in the albumin and alpha-globulin zones proved to be serum ones. An antigen with a mobility approximating 0 is specific for the kidney and liver. An antigen with a mobility in the alpha 2-globulin zone does not seem likely to be homogeneous and, apart from broad inter-organ specificity, bears the greatest resemblance to the lungs antigen.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Identification of multiple microtubule initiating sites in mouse neuroblastoma cells.

Mouse neuroblastoma N-18 cells can be induced by serum deprivation to sprout multiple neurite-like processes which contain many microtubules. Mitotic drugs such as colcemid and colchicine depolymerize these microtubules and the cells lose their processes. Reappearance of microtubules after removal of the drugs was followed by immunofluorescence microscopy using tubulin specific antibodies. At early recovery times multiple star-like structures which contained tubulin were detected in the perinuclear are and in the cytoplasm of individual cells. The mean number seen per cell as approximately 5. Their formation preceeded the organization of the complex microtubular networks typical of N-18 cells. The probable action of these structures as microtubular organization centers (MTOCs) is discussed. Multiple structures were detected during recovery from the influence of mitotic drugs both in previously induced and non-induced N-18 cells, suggesting that N-18 cells harbour the potential of formation of multiple organization centers even without previous induction. We discuss the possibility that differentiation of neuroblastoma N-18 cells may require microtubular organization centers.     

Cytoplasmic transfer of microtubule organizing centers in mouse tissue culture cells

A chloramphenicol-resistant, aminopterin-sensitive cell line (AMT) derived from a mouse mammary tumor MT-29240 was enucleated, and the cytoplasts were fused with nucleated chloramphenicol-sensitive, HAT-resistant SV40 3T3 mouse cells. The resulting cytoplasmic hybrids (cybrids) were selected for their resistance to chloramphenicol and the chromosome complement of the SV40 3T3 cells. In addition to transfer of chloramphenicol resistance, these cybrid clones, as studied in the electron microscope, contained the intracisternal A particle phenotype characteristic of only the AMT cells. The cytoplasmic microtubule complex (CMTC) in these cybrids was also studied and appears to resemble the elaborate CMTC of the AMT cells more closely than the more reduced CMTC of the SV40 3T3. When treated with a colcemid block and then a brief reverse, the microtubule organizing centers (MTOC) appear as bright fluorescent foci when tubulin antibody and indirect immunofluorescence techniques are used. When AMT or SV40 3T3 cells are treated in this manner, only one MTOC is present in interphase cells. One clone of these cybrids, however, contained two MTOCs in interphase cells. This CMTC and MTOC phenotype has persisted in this cybrid clone for over 3 months of continuous culture.     

Calcium transport in epithelial cells of the intestine and kidney

The central role of 1α,25-dihydroxyvitamin D₃ in the regulation of calcium balance is well established. By increasing the absorption of calcium in the intestine and the reabsorption of filtered calcium in the kidney tubule, the hormone maintains an appropriate calcium balance. The cellular mechanisms that underlie the increase in calcium transport in epithelial cells in response to 1α,25-dihydroxyvitamin D₃ are beginning to be defined. These events include an increase in the movement of calcium across the apical membrane of the cell, an increase in the movement of calcium across the cell, and an increase in the extrusion of calcium at the basolateral portion of the cell. In this Prospects article, I will discuss the nature of the various processes and proteins involved in transcellular calcium movement, and I will attempt to highlight various future areas of research.     

Trisomy 7 in non-neoplastic tubular epithelial cells of the kidney

The cellular origin of trisomy 7 in non-neoplastic kidney tissue specimens from 10 patients, seven with malignant tumors and three with non-neoplastic kidney diseases, was studied by the MAC (morphology antibody chromosomes) technique, which allows analysis of cellular morphology/histology, immunophenotype, and chromosomal aneuploidy by conventional cytogenetics, and/or fluorescent in situ hybridization in both interphase and mitotic cells. In primary cultures, trisomy 7 was detected primarily in cytokeratin-positive cells. Among freshly isolated renal cells, the trisomy was mainly observed in proximal tubular cells positive to brush-border antigen, and, to a lesser extent, in distal tubular cells positive to Tamm-Horsfall glycoprotein. The frequency of trisomy 7 in lymphocytes expressing CD3 or CD22 antigens isolated from non-neoplastic and tumor tissues was substantially lower than in the epithelial cells and was not increased compared with that in control lymphocytes from peripheral blood. The results thus demonstrate that the non-neoplastic kidney cells with trisomy 7 are mainly normal epithelial cells, preferentially those of the proximal tubule.     

Initiation and characterization of primary mouse kidney epithelial cultures

Summary Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃; c) high alkaline phosphatase activity; and d) Na⁺-dependent transport of phosphate (Pi) and α-methylglucoside (α-MG). The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems for Pi and α-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium. Phorbol esters and PTH did not exert an effect on Pi and α-MG transport in mouse primary cultures. The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function. Cindy Bell was the recipient of an MRC Studentship Award. This work was supported by the MRC (Group in Medical Genetics). This is publication number 88011 of the McGill University-Montreal Children's Hospital Research Institute.     

Intrinsic epithelial cells repair the kidney after injury

Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney.     

Establishment of immortal swine kidney epithelial cells

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.     

Role of lymphoid cells of the immune system in the genesis of kidney diseases]

Experiments on mice CBA X C57BL showed that the mixture of cells of the bone marrow, thymus, and spleen from donors with experimental hepatitis caused by carbon tetrachloride and from donors with resection of the normal liver, injected to healthy animals, caused dystrophic changes in the liver; this testifies to the presence of a special clone of cells with a damaging hepatotrophic effect, and participating in the mechanism of autoimmune disturbances in diseases of the liver.