Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough 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

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells

Determination of intracellular pH using sensitive, clickable fluorescent probes

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK a value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H +] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using \u27click\u27 chemistry. © 2011 Elsevier Ltd. All rights reserved

Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation

Structure and Biomechanics of the Endothelial Transcellular Circumferential Invasion Array in Tumor Invasion

<div><p>Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.</p></div