Tangential tether extraction and spontaneous tether retraction of human neutrophils

Abstract

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.

Figure 1

The microcantilever technique. (a) Schematic. (b) Cantilever calibration. Dotted lines show how the enlarged cantilever corresponds to the small rectangular area in a. The cantilever stiffness was calibrated by pushing it with a known force, imposed by the bead inside the micropipette. The cantilever deflection was tracked using the single-particle tracking algorithm (28).

Figure 2

Microscopic view of perpendicular (a) and tangential (b) tether extraction. The bead affixed on the cantilever tip was coated with antibodies against proteins on the cell, which was held by the micropipette. The cell was moved to contact the right side (a) or the top (b) of the bead, then pulled to the right to allow perpendicular (a) or tangential (b) tether extraction from the cell. If a tether was extracted, the cantilever would deflect to the right.

Figure 3

Tether-cell junction mobility in tangential and oblique tether extraction. Dotted circles represent the bead position when it was in contact with the cell. (a) After making contact with the top of the cell, the bead was moved to the left. (b) After making contact with the left side of the cell, the bead was moved briefly to the left and then upward. φ is the angle between the tether and the cell surface (φ = 0° and 90° for tangential and perpendicular tethers, respectively). θ is the angle defined by the tether-cell junction, the upper micropipette corner (the vertex of θ), and the cell center, so the change in θ represents the movement of the tether-cell junction. (c) Fourteen consecutive images (0.033 s between adjacent frames) showing a tangential tether being extracted from a neutrophil, only part of which is shown (the black cap). Fluorescent images are inverted for clarity herein and below. The membrane tether is shown as the faint black shadow emanating from the cell. Note that the bead is invisible. The black dotted line indicates the left end of the growing tether. The tether-cell junction stayed on the vertical white dotted line, showing its immobility. (d) A tether being extracted obliquely from a neutrophil. The black circle shows the position of the bead. (e) Quantification of the lateral mobility of the tether-cell junction on the cell surface for tethers extracted in different pulling directions (φ). The vertical arrow indicates the frame number at which the pulling bead started moving upward (refer to b). Obviously, the angle θ remained nearly constant, indicating that no sliding of the tether-cell junction occurred.

Figure 4

A typical force curve for tangential tether extraction. The approach, contact, and pull are marked, as are the single-tether extraction and detachment. The pulling speed was 25 μm/s.

Figure 5

The relationships between F and U_t for both perpendicular and tangential tether extraction. Each point represents an average of ∼50 tether extractions (error bars denote standard deviations).

Figure 6

Multiple tether visualization. No coalescence was observed when double tether extraction was performed. Only part of the cell is shown on the right.

Figure 7

Retraction of a tether extracted at 5 μm/s. (a) Tether length measured over time (circles). The solid curve is the best fit with the model described in the Appendix. (b) Video frames (0.033 s between adjacent frames) corresponding to the length measurements in a.