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Rhombic organization of microvilli domains found in a cell model of the human intestine.

ABSTRACT: Symmetry is rarely found on cellular surfaces. An exception is the brush border of microvilli, which are essential for the proper function of transport epithelia. In a healthy intestine, they appear densely packed as a 2D-hexagonal lattice. For in vitro testing of intestinal transport the cell line Caco-2 has been established. As reported by electron microscopy, their microvilli arrange primarily in clusters developing secondly into a 2D-hexagonal lattice. Here, atomic force microscopy (AFM) was employed under aqueous buffer conditions on Caco-2 cells, which were cultivated on permeable filter membranes for optimum differentiation. For analysis, the exact position of each microvillus was detected by computer vision; subsequent Fourier transformation yielded the type of 2D-lattice. It was confirmed, that Caco-2 cells can build a hexagonal lattice of microvilli and form clusters. Moreover, a second type of arrangement was discovered, namely a rhombic lattice, which appeared at sub-maximal densities of microvilli with (29 ± 4) microvilli / ?m2. Altogether, the findings indicate the existence of a yet undescribed pattern in cellular organization.

SUBMITTER: Franz J

PROVIDER: S-EPMC5761853 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Rhombic organization of microvilli domains found in a cell model of the human intestine.

Franz Jonas J Grünebaum Jonas J Schäfer Marcus M Mulac Dennis D Rehfeldt Florian F Langer Klaus K Kramer Armin A Riethmüller Christoph C

PloS one 20180110 1

Symmetry is rarely found on cellular surfaces. An exception is the brush border of microvilli, which are essential for the proper function of transport epithelia. In a healthy intestine, they appear densely packed as a 2D-hexagonal lattice. For in vitro testing of intestinal transport the cell line Caco-2 has been established. As reported by electron microscopy, their microvilli arrange primarily in clusters developing secondly into a 2D-hexagonal lattice. Here, atomic force microscopy (AFM) was ...[more]

PMID: 29320535

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Project description:Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.

Project description:Background and Purpose: Novobiocin (NOVO), an ABC transporter inhibitor, decreases intestinal wall permeability of capsaicin (CAP), an ABC transporter substrate. However, the mechanism of this effect is not consistent with the action of NOVO as an ABC transporter inhibitor. We previously found that CAP can also be transported via TRPV1, which was site-specific in the permeability of CAP across the intestine. We explored the regulation by NOVO of TRPV1 in the present study. Methods: Rats and transfected IEC-6 cells were used as the models to assess intestinal permeability and expression of TRPV1. Ussing chamber and intracellular accumulation were used to evaluate the influence of NOVO on the transport of CAP in vitro. The expression of TRPV1 was detected after administration of NOVO by qRT-PCR, western blot and immunofluorescent imaging. In addition, MTT and lactate dehydrogenase (LDH) were used to evaluate the cytotoxicity of NOVO in both rat and cell models. Finally, the effect of NOVO on the absorption of CAP in vivo was studied by LC-MS/MS. Results: In vitro data showed that there existed a dose-dependent relationship in the range of concentration between 5 and 50 μM, and even 5 μM NOVO could decrease intestinal permeability of CAP across the intestine. Meanwhile, cytosolic accumulation of CAP decreased when NOVO was used simultaneously or 24 h in advance. NOVO exhibited an inhibition level similar to that of ruthenium red (RR) or SB-705498, a TRPV1-specific inhibitor. NOVO down-regulated TRPV1 expression in the intestine and in transfected cells in a concentration-dependent fashion, hinting that its inhibition of the permeability of CAP is due to its inhibition of TRPV1 expression. Immunofluorescent imaging data showed that the fluorescence intensity of TRPV1 was reduced after pre-treatment with NOVO and SB-705498. In vivo data further demonstrated that oral co-administration of NOVO decreased Cmax and AUC of CAP in dosage-dependent ways, consistent with its role as a TRPV1 inhibitor. Conclusion: NOVO could be a potential TRPV1 inhibitor by attenuating the expression of TRPV1 and may be used to attenuate permeability of TRPV1 substrates.

Dataset Information

Rhombic organization of microvilli domains found in a cell model of the human intestine.

Publications

Rhombic organization of microvilli domains found in a cell model of the human intestine.

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