Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Arts, Janine; Mahlandt, Eike K.; Grönloh, Max L.B.; Schimmel, Lilian; Noordstra, Ivar; Steen, Abraham C I van; Tol, Simon; Rijssel, Jos van; Nolte, Martijn A.; Postma, Marten; Khuon, Satya; Heddleston, John M.; Wait, Eric; Chew, Teng‐Leong; Winter, Mark; Montañez, Eloi; Goedhart, Joachim; Buul, Jaap D. van

doi:10.1101/2021.01.18.427135

Cited by 3 publications

(3 citation statements)

References 61 publications

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“…A coverage estimate of the JMP by PECAM or VE-Cadherin stains is also computed by considering the distribution of staining across the JMP regions (details in the supplemental methods). This pipeline was written in MATLAB R2019b (source code can be found on Zenodo at https://doi.org/10.5281/zenodo.5494546 ; Arts et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Arts

Mahlandt

Grönloh

et al. 2021

eLife

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Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

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Section: Methodsmentioning

confidence: 99%

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Arts

Mahlandt

Grönloh

et al. 2021

eLife

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“…We have recently explored another way to visualize dynamics in data acquired by timelapse imaging [8]. In this case, we wanted to quantify how intensities of fluorescence signals changes over time by determining the coefficient of variation (standard deviation divided by the mean) on a pixel-by-pixel basis.…”

Section: Methodsmentioning

confidence: 99%

“…In this case, we wanted to quantify how intensities of fluorescence signals changes over time by determining the coefficient of variation (standard deviation divided by the mean) on a pixel-by-pixel basis. The imaging data was acquired from endothelial cells of which the plasma membrane was labeled with the fluorescent protein mNeongreen tagged with a CaaX box [8]. - Open the sequence ‘mNeon-CAAX.tif’ - Any other sources of fluctuations or changes in intensity are corrected for.…”

Section: Methodsmentioning

confidence: 99%

Visualizing and quantifying data from timelapse imaging experiments

Goedhart

2021

Preprint

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One obvious feature of life is that it is highly dynamic. The dynamics can be captured by movies that are made by acquiring images at regular time intervals, a method that is also known as timelapse imaging. Looking at movies is a great way to learn more about the dynamics in cells, tissue and organisms. However, science is different from Netflix, in that it aims for a quantitative understanding of the dynamics. The quantification is important for the comparison of dynamics and to study effects of perturbations. Here, we provide detailed processing and analysis methods that we commonly use to analyze and visualize our timelapse imaging data. All methods use freely available open-source software and use example data that is available from an online data repository. The step-by-step guides together with example data allow for fully reproducible workflows that can be modified and adjusted to visualize and quantify other data from timelapse imaging experiments.

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Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

Mahlandt

Martínez

Arts

et al. 2022

Preprint

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The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated to allow the passage of essential molecules like oxygen, carbon-dioxide, water, ions, and nutrients. The vascular endothelial barrier is defined by cell-cell contacts through adherens and tight junctions. To further investigate the signaling in the endothelium that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. Rho GTPase signaling is confined in space and time. To manipulate the signaling in a temporal and spatial manner we applied optogenetics. Guanine exchange factor (GEF) domains from ITSN1, TIAM1 and p63RhoGEF, activating Cdc42, Rac and Rho respectively, were integrated into the optogenetic recruitment tool iLID. This tool allows for activation at the subcellular level in a reversible and non-invasive manner and thereby to recruit a GEF to local areas at the plasma membrane, enabling the local activation of specific Rho GTPases. The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting Opto-RhoGEFs were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell-size, -roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool i.e. Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

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Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Cited by 3 publications

References 61 publications

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Visualizing and quantifying data from timelapse imaging experiments

Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

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