Role of microtubules in milk secretion--action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells

Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.     

Defensins are mitogenic for epithelial cells and fibroblasts

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth-promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.     

Difference in antigen presentation pathways between cortical and medullary thymic epithelial cells

Antigen presentation by thymic epithelial cells (TEC) to T cells that undergo maturation is one of the major events in the selection of the T cell repertoire. We have already reported that medullary TEC lines (mTEC) established from newborn C57BL/6 (H-2b) mice are able to present a soluble antigen, ovalbumin (OVA), to OVA-specific, I-Ab restricted helper T cell lines but cortical TEC (cTEC) lines are not (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601). In this report, to clarify the cause of this difference, we analyzed the biochemical nature as well as the distribution of both major histocompatibility complex (MHC) class II molecules and invariant chains (Ii) in both TEC by immunoprecipitation and laser confocal scanning microscopic analysis, as well as the expression of mRNA encoding H-2Ma or H-2Mb. Our results demonstrate that cTEC and mTEC are both able to present peptide antigens to peptide-specific, I-Ab-restricted helper T cell hybridoma and are able to present class II MHC alloantigens to an I-Ab-specific T cell line, that mRNA for H-2Ma and H-2Mb are expressed in both TEC, that cTEC and mTEC apparently incorporate tetramethylrhodamine isothiocyanate-labeled OVA in the same manner, and that the SDS-stable MHC class II molecules, onto which peptides were loaded, are formed in both cTEC and mTEC. However, these molecules were more rapidly degraded in mTEC than in cTEC. In addition, two Ii-derived polypeptides of approximately 21 kDa and 10 kDa were precipitated by the anti-class II monoclonal antibody Y3P; 10-kDa polypeptides were detected in the both TEC, while 21-kDa polypeptides were detected only in cTEC. Finally, beta chains of MHC class II with less sialylated oligosaccharides were precipitated from the cell surface of cTEC. Taken together, these results suggest that there are substantial differences in the antigen-presenting pathways of cTEC and mTEC, and these difference might be responsible for T cell selection events in the thymus.     

miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis

When neuroblasts divide, prospero protein and mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. miranda is known to localize prospero protein to the basal cell cortex of neuroblasts while the staufen RNA-binding protein mediates prospero mRNA localization. Here we show that miranda is required for asymmetric staufen localization in neuroblasts. Analyses using miranda mutants reveal that prospero and staufen interact with miranda under the same cell-cycle-dependent control. miranda thus acts to partition both prospero protein and mRNA. Furthermore, miranda localizes prospero and staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Our observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry.     

Endoplasmic reticulum membrane tubules are distributed by microtubules in living cells using three distinct mechanisms

BACKGROUND: The microtubule-dependent motility of endoplasmic reticulum (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have arisen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movement mechanism' in which ER attaches to moving microtubules; and the 'tip attachment complex (TAC) mechanism' in which ER tubules attach to growing plus ends of microtubules. RESULTS: We have used multi-wavelength time-lapse epifluorescence microscopy to image the dynamic interactions between microtubules (by microinjection of X-rhodamine-labeled tubulin) and ER (by DiOC6(3) staining) in living cells to determine which mechanism contributes to the formation and motility of ER tubules in migrating cells in vivo. Newly forming ER tubules extended only in a microtubule plus-end direction towards the cell periphery: 31.4% by TACs and 68.6% by the membrane sliding mechanism. ER tubules, statically attached to microtubules, moved towards the cell center with microtubules through actomyosin-based retrograde flow. TACs did not change microtubule growth and shortening velocities, but reduced transitions between these states. Treatment of cells with 100 nM nocodazole to inhibit plus-end microtubule dynamics demonstrated that TAC motility required microtubule assembly dynamics, whereas membrane sliding and retrograde-flow-driven ER motility did not. CONCLUSIONS: Both plus-end-directed membrane sliding and TAC mechanisms make significant contributions to the motility of ER towards the periphery of living cells, whereas ER removal from the lamella is powered by actomyosin-based retrograde flow of microtubules with ER attached as cargo. TACs in the ER modulate plus-end microtubule dynamics.     

Linear antigenic sites defined by the B-cell response to human p53 are localized predominantly in the amino and carboxy-termini of the protein

Using a set of overlapping peptides of the human p53 protein, we analysed the epitopes recognized by 18 monoclonal antibodies specific for human p53. We showed that most of these epitopes correspond to linear antigenic determinants which lie predominantly in the amino- or carboxy-terminus of the p53 protein. Using either truncated p53 or the set of human p53 peptides, we directly analysed the sera of animals immunized with human p53. These sera contained antibodies which also recognized the regions corresponding to the extremity of the p53 protein. These p53 regions were similar to those recognized by p53-specific antibodies present in sera of patients with cancer. Preferential recognition of these regions by antibodies specific for non conformational epitopes suggested that these regions are localized at the surface of the p53 protein as unfolded structures.     

Disruption of microtubules reveals two independent apical targeting mechanisms for G-protein-coupled receptors in polarized renal epithelial cells

G-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The alpha2A adrenergic receptor (alpha2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the alpha2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340-11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995-1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017-5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of alpha2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of alpha2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or alpha2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms.     

Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed

Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.     

Age differences in dual-task interference are localized to response-generation processes.

Dual-task differences in younger and older adults were explored by presenting 2 simple tasks, with the onset of the 2nd task relative to the 1st task carefully controlled. The possibility of an age-related reduction in the ability to generate and execute 2 similar motor programs was explored by requiring either a manual response to both tasks or a manual response to the 1st and an oral response to the 2nd and was confirmed by the evidence. The age-related interference was greater than would be expected from a general slowing of processing in older adults. The possibility of an age-related reduction in the capacity to process 2 tasks in the same perceptual input modality was explored by presenting both tasks in the visual modality or the 1st task in the auditory modality and the 2nd task in the visual modality and was not supported by the evidence. There was greater interference when both tasks were in the same modality, but it was equivalent for older and younger adults. Age differences in dual-task interference appear quite localized to response-generation processes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.     

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked "hyperpolarization," i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.     

Expression of telomerase activity in human endometrium is localized to epithelial glandular cells and regulated in a menstrual phase-dependent manner correlated with cell proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

Peptide-amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla

C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine alpha-amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology.     

Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells

The purpose of the present work was to test the hypothesis that no more than one vesicle of transmitter can be liberated by an action potential at a single release site. Spontaneous and evoked IPSCs were recorded from interneurons in the molecular layer of cerebellar slices. Evoked IPSCs were obtained using either extracellular stimulation or paired recordings of presynaptic and postsynaptic neurons. Connections were identified as single-site synapses when evoked current amplitudes could be grouped into one peak that was well separated from the background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing the release probability by lowering the external Ca2+ concentration or adding Cd2+ failed to reveal smaller quantal components. Some spontaneous IPSCs (1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were followed within <5 msec by a secondary IPSC that could not be accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after single release events. At some single-site connections, two closely spaced amplitude peaks were resolved, presumably reflecting single and double vesicular release. Consistent with multivesicular release, kinetics of onset, decay, and latency were correlated to IPSC amplitude. We conclude that the one-site, one-vesicle hypothesis does not hold at interneuron-interneuron synapses.     

Multiple attachment mechanisms of corneal epithelial cells to a polymer--cells can attach in the absence of exogenous adhesion proteins through a mechanism that requires microtubules

STUDY OBJECTIVE: To determine whether emergency patients with acute chest pain and low suspicion of acute myocardial infarction (AMI) can be managed cost-effectively and safely in a dedicated chest pain center (CPC) that incorporates mandatory stress testing. METHODS: We assembled a prospective observational case series of consecutive adult patients transferred from the emergency department to a nine-bed, 23-hour CPC in a 564-bed community hospital from January 13 through May 31, 1994. In our institution, all emergency patients with acute nontraumatic chest pain of unclear origin, suggestive of myocardial ischemia but with a low probability of AMI, are transferred to the CPC for further evaluation. All patients in whom AMI is ruled out undergo individually appropriate cardiac diagnostic testing in accordance with CPC clinical guidelines. Patients with end-stage coronary artery disease transferred to the CPC for a "rule-out" protocol only did not undergo further diagnostic testing. Admitted and discharged patients were followed through chart review and telephone survey, respectively. RESULTS: Of the 502 patients transferred to the CPC, 477 (95%) completed follow-up at 14 days. Four hundred ten (86%) were discharged home. Those discharged after diagnostic evaluation yielded negative findings had 100% survival and zero diagnosis of AMI at 5-month follow-up. Overall mortality and incidence of AMI on long-term follow-up for all patients transferred to the CPC were .4% and .2%, respectively. Sixty-seven patients (13%) were admitted from the CPC, of whom 44 (66%) had a final diagnosis of ischemic heart disease (IHD) or AMI. Twenty-four patients with IHD (55%; 6% of stress-tested group) were identified only on further stress testing. Of these patients, seven underwent percutaneous transluminal coronary angioplasty or coronary artery bypass grafting during hospitalization. All were discharged home without major morbidity. Four hundred twenty-four patients (84%) underwent stress testing. The cost of mandatory stress testing to identify one patient with IHD after AMI was ruled out was $3,125. An average cost-per-case savings of 62% was achieved for each patient transferred to the CPC who would have been hospitalized before the inception of the CPC. CONCLUSION: Mandatory stress testing is a safe, cost-effective, and valuable diagnostic and prognostic tool in CPC patients.     

Essential mitotic functions of DNA topoisomerase IIalpha are not adopted by topoisomerase IIbeta in human H69 cells

Unique functions of mammalian DNA-topoisomerases IIalpha and -beta are suggested by their distinct cellular distribution and chromatin binding at mitosis. Here, we studied H69-VP cells that, due to a homozygous mutation, express topoisomerase IIalpha mostly outside the nucleus. In these cells topoisomerase IIbeta showed a normal nuclear localization. However, at mitosis it diffused away from the chromatin despite the nuclear lack of the alpha-isoform. 80% of these cells performed chromosome condensation and disjunction with the aid of cytosolic topoisomerase IIalpha, which bound to the mitotic chromatin with low affinity. However, the genotype of these cells was highly polyploid indicating an increased rate of non-disjunction. In 20% of the mutant cells neither topoisomerase II isoform was bound to the mitotic chromatin, which appeared as an unstructured DNA spheroid unable to undergo disjunction and cytokinesis. Parental H69 cells expressing topoisomerase IIalpha inside the nucleus exhibited high affinity binding of the enzyme to the mitotic chromatin. Their genotype was mostly diploid and stable. We conclude (i) that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and (ii) that topoisomerase IIbeta does not adopt these functions.     

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis

PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.