Dataset Information

Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

ABSTRACT: Surface contamination and the formation of water bridge at the nanoscopic contact between an atomic force microscope tip and cell surface limits the maximum achievable spatial resolution on cells under ambient conditions. Structural information from fixed intestinal epithelial cell membrane is enhanced by fabricating a silicone liquid membrane that prevents ambient contaminants and accumulation of water at the interface between the cell membrane and the tip of an atomic force microscope. The clean and stable experimental platform permits the visualisation of the structure and orientation of microvilli present at the apical cell membrane under standard laboratory conditions together with registering subcellular details within a microvillus. The method developed here can be implemented for preserving and imaging contaminant-free morphology of fixed cells which is central for both fundamental studies in cell biology and in the emerging field of digital pathology.

SUBMITTER: Nirmalraj P

PROVIDER: S-EPMC6050225 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

Nirmalraj Peter P Lehner Roman R Thompson Damien D Rothen-Rutishauser Barbara B Mayer Michael M

Scientific reports 20180717 1

Surface contamination and the formation of water bridge at the nanoscopic contact between an atomic force microscope tip and cell surface limits the maximum achievable spatial resolution on cells under ambient conditions. Structural information from fixed intestinal epithelial cell membrane is enhanced by fabricating a silicone liquid membrane that prevents ambient contaminants and accumulation of water at the interface between the cell membrane and the tip of an atomic force microscope. The cle ...[more]

PMID: 30018393

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Project description:An improved oxygen availability in air-liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-D-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

Project description:The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.

Dataset Information

Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

Publications

Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

Similar Datasets

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