Jin-Yu Shao

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.

Mechanical Anchoring Strength of L-Selectin, β2 Integrins, and CD45 to Neutrophil Cytoskeleton and Membrane

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.

A Modified Micropipette Aspiration Technique and Its Application to Tether Formation From Human Neutrophils

Tether formation, which is mechanically characterized by its threshold force and effective viscosity, is involved in neutrophil emigration from blood circulation. Using the micropipette aspiration technique, which was improved by quantitative contact control and computerized data analysis, we extracted tethers from human neutrophils treated with IL-8, PMA, or cytochalasin D. We found that both IL-8 and PMA elevated the threshold force to about twice as large as the value for passive neutrophils. All these treatments decreased the effective viscosity dramatically (approximately 80%). With a novel method, the residual cortical tension of the cytochalasin-D-treated non-spherical neutrophils was measured to be approximately 8.8 pN/microm.

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of approximately 21 pN and approximately 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.

Quantifying cell-adhesion strength with micropipette manipulation: principle and application.

Quantifying cell-adhesion strength is of great importance in biology and medicine. Cell-adhesion strength can be characterized by separating two adherent cells and determining the force required to do so, or by measuring the lifetime of a receptor-ligand bond that mediates cell adhesion. To this end, several micropipette-based experimental techniques that operate at both cellular and molecular levels have been developed over the past few decades. In this review, we provide an overview of three of these techniques, i.e., the step-pressure technique (SPT), the biomembrane-force probe (BFP), and the micropipette-aspiration technique (MAT). More detailed discussion will be given about the requirements and applications of the MAT.

The Resistance to Flow of Individual Human Neutrophils in Glass Capillary Tubes with Diameters between 4.65 and 7.75 μm

Objective: The resistance to flow of individual human neutrophils flowing through smooth‐walled glass capillary tubes at velocities ranging between 0 and 30 μm/s is measured for six different capillary tube diameters between 4.65 and 7.75 μm.

A Model for CD2/CD58-Mediated Adhesion Strengthening

Stable cell adhesion is vital for structural integrity and functional efficacy. Yet how low affinity adhesion molecules such as CD2 and CD58 can produce stable cell adhesion is still not completely understood. In this paper, we present a theoretical model that simulates the accumulation of CD2 and CD58 in the contact area of a Jurkat T lymphoblast and a CD58-containing substrate. The cell is assumed to have a spherical shape initially and it is allowed to spread gradually on a circular substrate. Mobile CD2 and CD58 can diffuse freely on both the cell and substrate. Their binding in the contact area is controlled by first-order kinetics. The contact area grows linearly with the total number of CD2/CD58 bonds. Cellular deformation and cytoskeleton involvement were not considered. This time-dependent moving-boundary problem was solved with the Crank–Nicolson finite difference scheme and the variable space grid method. Our simulated results are in reasonable agreement with the experimental observations. The role of diffusion becomes more and more prominent during the contact area increase, which is not sensitive to the kinetic rate constants tested in this study. However, it is very sensitive to the dissociation equilibrium constant and the concentrations of CD2 and CD58.

Finite element analysis of imposing femtonewton forces with micropipette aspiration.

AbstractA novel technique of imposing femtonewton forces with micropipette aspiration [i.e., the extended micropipette aspiration technique (EMAT)] is proposed, and an axisymmetric finite element analysis of this technique is provided. The EMAT is experimentally based upon a micropipette manipulation system and is theoretically based upon hydrodynamics. Any spherical object such as a human neutrophil or a latex bead can be employed as the force transducer, so cell–cell interactions can be directly studied. Our computational analysis shows that femtonewton forces can indeed be imposed. The force magnitude is sensitive to the radius of the micropipette and the micropipette-transducer distance, but it is much less sensitive to other parameters including the radius of the transducer, the substrate curvature, and the thickness of the micropipette wall. Combining the EMAT and the previously developed micropipette aspiration technique will allow us to impose an unprecedented range of forces, from a few femtonewtons to a few hundred piconewtons on single molecules or receptor-ligand bonds.

Tangential Tether Extraction and Spontaneous Tether Retraction of Human Neutrophils

Membrane tethers are extracted when neutrophils roll on the endothelium to initiate their transendothelial migration. Tether extraction from both neutrophils and endothelial cells stabilizes neutrophil rolling, so it has been studied extensively and the force-velocity relationship for tether extraction is of great interest. Due to limitations of the techniques used in previous studies, this relationship has been obtained only from tethers perpendicular to the cell surface. Here, with the microcantilever technique, where latex beads affixed on silicon cantilevers were used as the force transducer, we extracted tethers either perpendicular or tangential to the neutrophil surface. We found that the force-velocity relationship was not sensitive to tether pulling direction. Little movement of the tether-cell junction was observed during tangential tether extraction, and no coalescence was observed during multiple tether extraction. Following adhesion rupture, spontaneous tether retraction was visualized by membrane staining, which revealed two phases: one was fast and exponential, whereas the other was slow and linear. Both phases can be reproduced with a mechanical model. These results show for the first time, to our knowledge, how neutrophil tethers shorten upon instantaneous force removal, and furthermore, they illustrate how membrane tethers contribute to neutrophil rolling stability during the inflammatory response.

A direct micropipette-based calibration method for atomic force microscope cantilevers

In this report, we describe a direct method for calibrating atomic force microscope (AFM) cantilevers with the micropipette aspiration technique (MAT). A closely fitting polystyrene bead inside a micropipette is driven by precisely controlled hydrostatic pressures to apply known loads on the sharp tip of AFM cantilevers, thus providing a calibration at the most functionally relevant position. The new method is capable of calibrating cantilevers with spring constants ranging from 0.01 to hundreds of newtons per meter. Under appropriate loading conditions, this new method yields measurement accuracy and precision both within 10%, with higher performance for softer cantilevers. Furthermore, this method may greatly enhance the accuracy and precision of calibration for colloidal probes.