scholarly journals The actin cytoskeleton governs apical mitosis and daughter cell dispersion in intestinal epithelia

2017 ◽  
Author(s):  
Kara L. McKinley ◽  
Nico Stuurman ◽  
Ophir D. Klein ◽  
Ronald D. Vale
Keyword(s):  
Actin Cytoskeleton ◽  
Apical Cell ◽  
Intestinal Epithelial ◽  
Interphase Nuclei ◽  
Cell Lineages ◽  
Intestinal Epithelia ◽  
Cell Dispersion ◽  
Dividing Cells ◽  
Shape Changes ◽  
Mitotic Behavior

AbstractCell proliferation is critical for maintaining the absorptive, protective and regenerative functions of the small intestine throughout adulthood. Interphase nuclei are positioned near the basal surface of the intestinal epithelium, but during mitosis, chromosomes are located apically. The molecular basis for apical-basal DNA positioning and its consequences for tissue homeostasis are poorly understood. Here, we image and pharmacologically perturb these behaviors in live murine intestinal organoids. We find that apical and basal DNA movements occur as a result of mitosis-coupled actin rearrangements that alter the basolateral shape of dividing cells, while the apical cell surface remains confined by cell-cell contacts that persist throughout mitosis. Strikingly, these polarized shape changes allow neighboring cells to insert between nascent daughters, intermingling cells of different lineages. In summary, polarized rearrangements of the actin cytoskeleton govern the mitotic behavior of intestinal epithelial cells and lead to interspersion of cell lineages.

Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 initiate signalling pathways involved in intestinal epithelial barrier protection

2018 ◽  
Vol 9 (3) ◽  
pp. 515-525 ◽  
Author(s):  
D. Paveljšek ◽  
P. Juvan ◽  
R. Košir ◽  
D. Rozman ◽  
B. Hacin ◽  
...  
Keyword(s):  
Growth Factor ◽  
Actin Cytoskeleton ◽  
Lactobacillus Fermentum ◽  
Signalling Pathways ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Apoptotic Pathway ◽  
Promising Alternative ◽  
Bifidobacterium Animalis

The manipulation of intestinal microbiota with beneficial microbes represents a promising alternative or adjunct therapy in gastrointestinal disorders and inflammation. The current study aims to clarify the signalling pathways and evaluate the possible beneficial effects of the combination of two strains. We used a dextran sulphate sodium (DSS)-induced mouse model of colitis. RNA extracted from the middle part of the colon tissue was used for examination of the global gene expression with Affymetrix microarrays. An enrichment analysis of the KEGG pathways was performed, and a subset of genes associated with intestinal epithelial barrier function was verified with qPCR. A clinical condition assessment of the differently treated mice revealed that the combination of these two bacterial strains was safe for use as a dietary supplement. All animals treated with DSS had affected colons and suffered weight loss. There were very small differences between the diseased groups, although the depth of inflammation was lower when cyclosporine A or the strain mixture was used. We discovered that the prophylactic administration of the Lactobacillus fermentum L930BB (L930BB) and Bifidobacterium animalis subsp. animalis IM386 (IM386) strains led to an anti-apoptotic pathway through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and to the activation of pathways involved in the regulation of actin cytoskeleton via protein kinase C and GTPases. Reorganisation of actin cytoskeleton and decreased apoptosis are both helpful in intestinal epithelial cell reconstitution. We confirm important previous observations, showing that these pathways are downstream targets of Toll-like receptor 2 and fibroblast growth factor initiated signalling. Taken together, these results suggest that the combination of L930BB and IM386 could aid in the regeneration of the intestinal epithelium during pathogenesis via pattern recognition receptors and the stimulation of growth factor synthesis.

Continuous DNA replication in the nucleus of the dinoflagellate Prorocentrum micans (Ehrenberg)

1977 ◽  
Vol 27 (1) ◽  
pp. 81-90
Author(s):  
S.A. Filfilan ◽  
D.C. Sigee
Keyword(s):  
Electron Microscope ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Continuous Process ◽  
Prorocentrum Micans ◽  
Interphase Nuclei ◽  
Electron Microscope Autoradiography ◽  
Dividing Cells ◽  
High Degree ◽  
Very High

The uptake of tritiated thymine into cells of a heterogeneous population of Prorocentrum micans was investigated using light-microscope and electron-microscope autoradiography. Specificity of thymine uptake into DNA was demonstrated by the specific removal of label from wax-embedded material using DNase and by the high degree of localization of nuclear label to chromosomes in the electron-microscope autoradiographs. All nuclei, including both dividing and non-dividing cells, showed a substantial uptake of label, indicating that nuclear DNA synthesis in Prorocentrum micans is a continuous process. The level of DNA synthesis does show considerable variation, however, with very high levels in some interphase nuclei. The continuous replication of nuclear DNA provides further evidence of dinoflagellate affinity to the prokaryotes, and indicates that Prorocentrum micans is a very primitive eukaryote cell.

scholarly journals Uptake and intracellular fate of l-DOPA in a human intestinal epithelial cell line: Caco-2

1998 ◽  
Vol 275 (1) ◽  
pp. C104-C112 ◽  
Author(s):  
M. A. Vieira-Coelho ◽  
P. Soares-Da-Silva
Keyword(s):  
Apical Cell ◽  
Acid Decarboxylase ◽  
Epithelial Cell Line ◽  
Uptake System ◽  
Intestinal Epithelial ◽  
Cell Border ◽  
Amino Acid Decarboxylase ◽  
Dopa Concentration ◽  
Menten Constant ◽  
Cells Cultured

The aim of the present study was to examine the kinetic characteristics of thel-3,4-dihydroxyphenylalanine (l-DOPA) transporter and the fate of newly formed dopamine in Caco-2 cells. In the presence of 50 μM benserazide (an inhibitor of aromaticl-amino acid decarboxylase), l-DOPA was rapidly accumulated in Caco-2 cells. At equilibrium (30 min of incubation) the intracellular l-DOPA concentration was 10.2 ± 0.1 μM at a medium concentration of 0.5 μM. In saturation experiments the accumulation of l-DOPA was saturable with a Michaelis-Menten constant ( K m) of 60 ± 10 μM and a maximal reaction velocity ( V max) of 6.6 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1; at 4°C the amount of l-DOPA accumulated in the cells was nonsaturable. When cells were incubated with increasing concentrations of l-DOPA (10–100 μM) in the absence of benserazide, a substantial amount of thel-DOPA that was taken up was decarboxylated to dopamine, with an apparent K m of 27.2 μM. In experiments performed in cells cultured in polycarbonate filters, the accumulation of l-DOPA in the presence of benserazide was greater when the substrate was applied from the basolateral cell border than when it was applied from the apical cell border. In the absence of benserazide, l-DOPA applied from the basolateral cell border resulted in a nonlinear formation of dopamine ( K m = 43 ± 7 μM, V max = 23.7 ± 1.2 nmol ⋅ mg protein−1 ⋅ 6 min−1). The amount of dopamine leaving the cell through the apical cell border was lower than the amount that escaped through the basolateral cell border, and the process was saturable ( K m = 623 ± 238 μM, V max = 0.19 ± 0.02 nmol ⋅ mg protein−1 ⋅ 6 min−1). In conclusion, the data presented here show that Caco-2 cells are endowed with an efficient l-DOPA uptake system, and intracellularl-DOPA was found to be rapidly converted to dopamine, some of which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way to look at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

Functional coupling of tight junctions and microfilaments in T84 monolayers

1988 ◽  
Vol 254 (3) ◽  
pp. G416-G423 ◽  
Author(s):  
J. L. Madara ◽  
J. Stafford ◽  
D. Barenberg ◽  
S. Carlson
Keyword(s):  
Tight Junction ◽  
Actin Binding ◽  
Functional Coupling ◽  
Flux Analysis ◽  
Intestinal Epithelial ◽  
Binding Agent ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Tight Junction Permeability ◽  
Cd Exposure

The actin-binding agent cytochalasin D (CD) in intact intestinal epithelium appears to elicit segmentation and contraction of a perijunctional ring of actomyosin and, consequently, to diminish tight junction resistance. We determined if an intestinal epithelial model composed of T84 cells also displayed such a perijunctional cytoskeletal specialization and, if so, whether exposure to CD also affected the tight junction barrier. We find T84 cells display a prominent perijunctional microfilament ring that is actin rich. CD elicits large decreases in transepithelial resistance due specifically to perturbed tight junction permeability as determined with dual Na+-mannitol flux analysis. Transepithelial mannitol and insulin fluxes also increase after CD exposure, but these molecules remain differentially restricted in accordance with their sizes, indicating that gross disruption of the monolayer has not occurred. Structurally, CD elicits segmentation and condensation of the perijunctional ring into actin-rich plaques. These features have similarity to those seen in native intestinal epithelia. This system may represent a simple model for studies of cytoskeletal-tight junction relationships.

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

Comparative analysis of ontogenic changes in renal and intestinal biotin transport in the rat

2003 ◽  
Vol 284 (4) ◽  
pp. F737-F742 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Veedamali S. Subramanian ◽  
Hamid M. Said
Keyword(s):  
Age Groups ◽  
Membrane Vesicles ◽  
Transport Processes ◽  
Water Soluble ◽  
Kidney Cortex ◽  
Intestinal Epithelial ◽  
Adult Rats ◽  
Intestinal Epithelia ◽  
Sodium Dependent ◽  
Uptake Process

Biotin, an essential water-soluble micronutrient, cannot be synthesized by mammals; rather, it is obtained from exogenous sources via uptake by intestinal epithelia. Renal epithelia reclaim the vitamin that is filtered in the glomeruli. Both epithelia take up biotin via the sodium-dependent multivitamin transporter (SMVT). Little is known about ontogenic regulation of the renal and intestinal biotin transport processes and about the mechanism(s) involved in any such regulation. In this study, we sought to examine and compare ontogenic aspects of the renal and intestinal biotin uptake processes using purified brush-border membrane vesicles (BBMV) isolated from the kidney cortex and jejunum of suckling and adult rats. Clear ontogenic changes were observed in the intestinal biotin uptake process, which were mediated via changes in V max and apparent K m. Parallel changes were also seen in protein, mRNA, and transcription rate of SMVT as indicated by results of Western blotting, RT-PCR, and nuclear run-on assays, respectively. In contrast, biotin uptake by renal BBMV did not show ontogenic changes; i.e., it was similar in suckling and adult rats. Also, the levels of SMVT protein and mRNA were similar in the kidneys of both age groups. These data show that biotin uptake by renal and intestinal epithelial cells responds differently to ontogenic regulation. In addition, the ontogenic changes observed in the intestinal biotin uptake process involve the entry step of the vitamin at the BBM and appear to be mediated via a transcriptional mechanism(s).

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia

2009 ◽  
Vol 296 (6) ◽  
pp. G1332-G1343 ◽  
Author(s):  
Annabelle Cesaro ◽  
Abakar Abakar-Mahamat ◽  
Patrick Brest ◽  
Sandra Lassalle ◽  
Eric Selva ◽  
...  
Keyword(s):  
High Activity ◽  
Early Phase ◽  
Active Form ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Tnf Α ◽  
Vitro Model

The acute phase of Crohn's disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-α. Conversion of inactive TNF-α into an active form requires the cleavage of a transmembrane TNF-α precursor by the TNF-α-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-α induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H2O2, or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.

scholarly journals LGN regulates mitotic spindle orientation during epithelial morphogenesis

2010 ◽  
Vol 189 (2) ◽  
pp. 275-288 ◽  
Author(s):  
Zhen Zheng ◽  
Huabin Zhu ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Zhuoni Xiao ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mdck Cells ◽  
Apical Cell ◽  
Cell Polarization ◽  
Epithelial Morphogenesis ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Dividing Cells ◽  
Cortical Localization ◽  
Lateral Cell

Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Gα-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Gα proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarization–mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.

Corticosteroid receptors and 11β-hydroxysteroid dehydrogenase isoforms in rat intestinal epithelia

1999 ◽  
Vol 277 (3) ◽  
pp. G541-G547 ◽  
Author(s):  
Karen E. Sheppard ◽  
Kevin X. Z. Li ◽  
Dominic J. Autelitano
Keyword(s):  
Enzyme Activity ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Reductase Activity ◽  
Distal Colon ◽  
Hydroxysteroid Dehydrogenase ◽  
Intestinal Epithelial ◽  
Rnase Protection ◽  
Intestinal Epithelia ◽  
Corticosteroid Receptors

To evaluate the potential roles that both receptors and enzymes play in corticosteroid regulation of intestinal function, we have determined glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11β-hydroxysteroid dehydrogenase (11β-HSD) expression in intestinal epithelial cells. GR and MR mRNA and receptor binding were ubiquitously expressed in epithelial cells, with receptor levels higher in ileum and colon than jejunum and duodenum. RNase protection analysis showed that 11β-HSD1 was not expressed in intestinal epithelial cells, and enzyme activity studies detected no 11-reductase activity. 11β-HSD2 mRNA and protein were demonstrated in ileal and colonic epithelia; both MR and GR binding increased when enzyme activity was inhibited with carbenoxolone. Duodenal and jejunal epithelial cells showed very little 11β-HSD2 mRNA and undetectable 11β-HSD2 protein; despite minor (<7%) dehydrogenase activity in these cells, enzyme activity did not alter binding of corticosterone to either MR or GR. These findings demonstrate the ubiquitous but differential expression of MR and GR in intestinal epithelia and that 11β-HSD2 modulates corticosteroid binding to both MR and GR in ileum and proximal and distal colon but not in duodenum or jejunum.

Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages

1994 ◽  
Vol 266 (6) ◽  
pp. G987-G1003 ◽  
Author(s):  
P. Falk ◽  
K. A. Roth ◽  
J. I. Gordon
Keyword(s):  
Epithelial Cell ◽  
Transgenic Mice ◽  
Intestinal Epithelial Cell ◽  
Cell Lineage ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Intestinal Epithelial ◽  
Cell Lineages ◽  
Carbohydrate Specificities

We have used histochemical methods to survey the cellular patterns of binding of a panel of 45 lectins with well-defined carbohydrate specificities to sections prepared from various regions of the gastric-to-colonic axis of fetal, neonatal, and adult FVB/N mouse gut. The results suggest that lectins can be used as remarkably sensitive tools to describe the differentiation programs of gastric and intestinal epithelial cell lineages as a function of their position along the cephalocaudal axis of the gut and as a function of developmental stage. Studies of intestinal isografts and transgenic mice that express Simian virus-40 T antigen in enterocytes suggest that many of these cell lineage-specific and spatial patterns of glycoconjugate production can be established and maintained in the absence of exposure to luminal contents and in the presence of specific proliferative abnormalities. This lectin panel should be useful for operationally defining subpopulations of the principal gut epithelial cell lineages in normal strains of mice, for describing variations in gut epithelial cell differentiation programs in mutant and transgenic mice, and for recovering specific epithelial cell lineages or subpopulations.

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