A microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer

Thuenauer, Roland; Nicklaus, Simon; Frensch, Marco; Troendle, Kevin; Madl, Josef; Römer, Winfried

doi:10.1039/c7ra11943g

Cited by 6 publications

(4 citation statements)

References 29 publications

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“…To explain the abrogation of cell migration by LecB, we carried out live-cell imaging experiments with MDCK cells stably expressing the plasma membrane marker ML-GFP (GFP tagged with a Lyn-derived myristoylation motif) (45). ML-GFP allowed visualization of lamellipodia formed by migrating MDCK cells (Fig.…”

Section: Thuenauer Et Almentioning

confidence: 99%

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

Thuenauer

Landi

Trefzer

et al. 2020

mBio

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The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of ␤1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive ␤1-integrins and also of complete ␣3␤1-integrin-laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cellbasement membrane attachment in a LecB-dependent manner.

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Section: Thuenauer Et Almentioning

confidence: 99%

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

Thuenauer

Landi

Trefzer

et al. 2020

mBio

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“…Thereby, the arrival of the proteins at the plasma membrane could be detected in real‐time by recombination of both fragments. Mardin Darby canine kidney (MDCK) cells were used because they readily form epithelial monolayers 3,14,15 . Rhodopsin (rhod) 13 and p75 16 were used as markers for apical protein trafficking and the neural cell adhesion molecule (NCAM) 17 as basolateral marker.…”

Section: Resultsmentioning

confidence: 99%

“…Mardin Darby canine kidney (MDCK) cells were used because they readily form epithelial monolayers. 3,14,15 Rhodopsin (rhod) 13 and p75 16 were used as markers for apical protein trafficking and the neural cell adhesion molecule (NCAM) 17 as basolateral marker.…”

Section: Functional Principle Of Split Luficerasemediated Detection O...mentioning

confidence: 99%

Real‐time monitoring of cell surface protein arrival with split luciferases

Fischer,

Schatz,

Baaske

et al. 2023

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Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000‐fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.

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“…Thereby, the arrival of the proteins at the plasma membrane could be detected in real time by recombination of both fragments. Mardin Darby canine kidney (MDCK) cells were used because they readily form epithelial monolayers [3,14,15]. Rhodopsin (rhod) [13] and p75 [16] were used as markers for apical protein trafficking and the neural cell adhesion molecule (NCAM) [17] as basolateral marker.…”

Section: Functional Principle Of Split Luficerase-mediated Detection ...mentioning

confidence: 99%

Real-time monitoring of cell surface protein arrival with split luciferases

Fischer

Baaske

Römer

et al. 2023

Preprint

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Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, the sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as tag on the protein of interest and the second fragment as supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.

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A microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer

Abstract: We developed a microfluidic biochip that enables one to locally change the basolateral microenvironment of epithelial cells within a polarised monolayer.

Cited by 6 publications

References 29 publications

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

Real‐time monitoring of cell surface protein arrival with split luciferases

Real-time monitoring of cell surface protein arrival with split luciferases

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