Static and dynamic lengths of neutrophil microvilli

Abstract

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both approximately 0.3 microm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is </=34 pN (+/- 3 pN), the length of the microvillus will be extended; if the force is >61 pN (+/- 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of approximately 43 pN/microm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

Figure 1

A scanning electron micrograph showing tips of microvilli from a human neutrophil adhering to an anti-CD45-coated bead.

Figure 2

A micrograph showing an anti-CD45-coated bead about to be touched by a human neutrophil. At the beginning of every experiment for a neutrophil-bead pair, a known suction pressure is applied inside the pipette on the left. Then a positive pressure is superimposed so that the neutrophil can move toward the bead. After the cell and the bead make contact, the positive pressure is released. If the cell does not adhere to the bead, the cell will move away freely under the suction pressure; if the cell adheres to the bead, the cell will stay close to the bead or it will move away at a smaller velocity than its free motion velocity. After the cell detaches from the bead, the positive pressure is superimposed again and the same procedure is repeated.

Figure 3

Microvillus extension (A) and tether formation (B). (A) The motion of a neutrophil before and after it adhered to an anti-CD45-coated bead under a suction pressure of 0.5 pN/μm². The slope of the dotted line stands for the velocity of the same cell when it is moving freely inside the same micropipette under the same pressure. ΔD is the displacement of the neutrophil relative to the adhesion point as the neutrophil approaches the bead, adheres, moves back freely for ≈0.4 μm (the free motion rebound), and then moves at a diminishing velocity (the diminishing rebound). After adhesion, ΔD actually represents the change in the microvillus length because the tip of the microvillus is stationary on the bead. The static equilibrium force (U = 0) that causes the extension of the microvillus is calculated with F = πR_p², whose average is 34 pN (uncertainty 3 pN) at 0.5 pN/μm². Neutrophils that adhered to anti-CD162-coated beads have similar behavior under the same suction pressure. (B) The motion of a neutrophil before and after it has formed a membrane tether under a suction pressure of 1 pN/μm². Here, the slope of the dotted line also stands for the velocity of the same cell when it is moving freely inside the same micropipette under the same pressure. After adhering to the bead and moving back freely for ≈0.4 μm (the free motion rebound), the neutrophil, attached by its membrane tether, continues to move linearly, albeit at a smaller velocity. At this suction pressure, the average force calculated with Eq. 1 from all the measurements is 61 pN (uncertainty 5 pN).

Figure 4

Natural lengths of microvilli (A) and extended lengths of microvilli (B). Error bars stand for their SDs. The measurements for anti-CD162 are drawn intentionally at a pressure 0.02 pN/μm² less than the actual pressure so that their error bars can be differentiated from the ones for anti-CD45. (A) The lack of dependence of the free motion rebound length (L₀) upon the suction pressure. L₀ is considered to be the natural length of a microvillus and is directly measured from the tracking data as shown in Fig. 3A and Fig. 3B. The solid circle shows the average for all the measurements. The number of measurements at each pressure is 7, 8, 14, 15, 9, 9, and 9 (left to right). The number of measurements for anti-CD162 at each of the three lower pressures is six. (B) The dependence of the extended length (ΔL) upon the suction pressure. ΔL is obtained by fitting L = L_∞ − ΔLe^−t/t_c to the diminishing rebound of the extension curve shown in Fig. 3A. The number of measurements for anti-CD45 at each pressure is 7, 8, 14, 11, 6, 5, and 1 (left to right). The number of measurements for anti-CD162 at each of the three lower pressures is six.

Figure 5

Force balance on an adhered neutrophil mediated by a single attachment. L is the total length of the microvillus, l is the length of the moment arm, R is the radius of the cell (≈4.25 μm), F_b is the force on the adhesive bond, F_s is the force, and T_s is the torque imposed by the shear flow on the cell. Because the velocity of a rolling cell is much less than the velocity of a neutrally buoyant cell, the force and torque calculation for a static cell in a shear flow is generally used (9, 12, 21).

Figure 6

Bond force (F_b) decrease (A), moment arm (l) and tether length (L) increase (B) for a rolling neutrophil with a single attachment at a shear stress of 0.08 pN/μm². An initial length of 0.35 μm for L was used. (A) The bond force decreased 50% in only ≈0.2 s. Then it tends to steady-state very slowly. (B) The fastest increase in both tether length and moment arm happened before 0.2 s. Also, both moment arm and tether grow more than twice their original lengths in 1 s. But they will eventually tend to constant values.