Maria K. Pospieszalska

/`Slings/' enable neutrophil rolling at high shear

Most leukocytes can roll along the walls of venules at low shear stress (1 dyn cm−2), but neutrophils have the ability to roll at tenfold higher shear stress in microvessels in vivo. The mechanisms involved in this shear-resistant rolling are known to involve cell flattening and pulling of long membrane tethers at the rear. Here we show that these long tethers do not retract as postulated, but instead persist and appear as ‘slings’ at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro, where P-selectin-glycoprotein-ligand-1 (PSGL-1) is found in discrete sticky patches whereas LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The ‘step-wise peeling of slings’ is distinct from the ‘pulling of tethers’ reported previously. Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling

We introduce quantitative dynamic footprinting microscopy to resolve neutrophil rolling on P-selectin. We observed that the footprint of a rolling neutrophil was fourfold larger than previously thought, and that P-selectin–PSGL-1 bonds were relaxed at the leading edge of the rolling cell, compressed under the cell center, and stretched at the trailing edge. Each rolling neutrophil formed three to four long tethers that extended up to 16 μm behind the rolling cell.

Event‐Tracking Model of Adhesion Identifies Load‐Bearing Bonds in Rolling Leukocytes

Objectives: P‐selectin binding to P‐selectin glycoprotein ligand‐1 (PSGL)‐1 mediates leukocyte rolling under conditions of inflammation and injury. The aims of this study were to develop an efficient, high temporal resolution model for direct simulation of leukocyte rolling and conduct a study of load‐bearing bonds using the model.

Cell Protrusions and Tethers: A Unified Approach

Low pulling forces applied locally to cell surface membranes produce viscoelastic cell surface protrusions. As the force increases, the membrane can locally separate from the cytoskeleton and a tether forms. Tethers can grow to great lengths exceeding the cell diameter. The protrusion-to-tether transition is known as the crossover. Here we propose a unified approach to protrusions and tethers providing, to our knowledge, new insights into their biomechanics. We derive a necessary and sufficient condition for a crossover to occur, a formula for predicting the crossover time, conditions for a tether to establish a dynamic equilibrium (characterized by constant nonzero pulling force and tether extension rate), a general formula for the tether material after crossover, and a general modeling method for tether pulling experiments. We introduce two general protrusion parameters, the spring constant and effective viscosity, valid before and after crossover. Their first estimates for neutrophils are 50 pN μm(-1) and 9 pN s μm(-1), respectively. The tether elongation after crossover is described as elongation of a viscoelastic-like material with a nonlinearly decaying spring (NLDs-viscoelastic material). Our model correctly describes the results of the published protrusion and tether pulling experiments, suggesting that it is universally applicable to such experiments.

Chapter 11 Intravital Microscopic Investigation of Leukocyte Interactions with the Blood Vessel Wall

Intravital microscopy is a method to study the microcirculation in living tissues. Transillumination, oblique reflected light illumination, continuous and stroboscopic epifluorescence microscopy can be used to visualized specific cells and molecules. Intravital microscopy is further enhanced by the advent of laser scanning.spinning disk confocal and multi-photon microscopy. Recent advances include blood-perfused flow chambers and microfluidic devises for the study of blood cell interactions with molecularly defined substrates. This chapter focuses on the application of these techniques to study leukocyte interactions with the vascular wall and molecular surfaces.

Chapter 8 Modeling Leukocyte Rolling

Publisher Summary Computer modeling is a powerful tool, giving detailed insights into in vivo biological processes and in vitro experiments. Typically, only some of the data generated by a model, and in a reduced form, can be observed or measured by an experimentalist. Leukocyte rolling—a behavior commonly observed in inflammation—is mediated by a continuous series of molecular bonds between the cell and the substrate that rapidly form and dissociate. Molecular site densities and parameters—such as molecular lengths and reaction rates—have been experimentally measured or estimated from experiments. The leukocytes are viscoelastic bodies studded with viscoelastic microvilli. Some of their viscoelastic properties are accessible to experimental interrogation. This chapter discusses the need for modeling leukocyte rolling, summarizes the history of research in this field, and guides through the development process of a leukocyte rolling model, including model parameters, model cellular, molecular and environmental interaction rules, model algorithm, and model validation. Some rolling models use a direct approach, where key molecules, bonds, and cellular elements are tracked in time and space, whereas others use semianalytic, analytical, or agent-based modeling methods.