Filopodia act as phagocytic tentacles and pull with discrete steps and a load-dependent velocity

Abstract

Filopodia are thin, spike-like cell surface protrusions containing bundles of parallel actin filaments. So far, filopodial dynamics has mainly been studied in the context of cell motility on coverslip-adherent filopodia by using fluorescence and differential interference contrast (DIC) microscopy. In this study, we used an optical trap and interferometric particle tracking with nanometer precision to measure the three-dimensional dynamics of macrophage filopodia, which were not attached to flat surfaces. We found that filopodia act as cellular tentacles: a few seconds after binding to a particle, filopodia retract and pull the bound particle toward the cell. We observed F-actin-dependent stepwise retraction of filopodia with a mean step size of 36 nm, suggesting molecular motor activity during filopodial pulling. Remarkably, this intracellular stepping motion, which was measured at counteracting forces of up to 19 pN, was transmitted to the extracellular tracked particle via the filopodial F-actin bundle and the cell membrane. The pulling velocity depended strongly on the counteracting force and ranged between 600 nm/s at forces <1 pN and approximately 40 nm/s at forces >15 pN. This result provides an explanation of the significant differences in filopodial retraction velocities previously reported in the literature. The measured filopodial retraction force-velocity relationship is in agreement with a model for force-dependent multiple motor kinetics.

Fig. 1.

Filopodial and ruffle retraction preceding phagocytosis. (A) An IgG-coated bead in the optical trap (orange circle) is moved toward a filopodium of a J774 macrophage. Upon binding, the filopodium (arrowhead) retracts and pulls the bead toward the cell to initiate phagocytic uptake. (B) Two trapped beads are moved toward a membrane ruffle. Upon binding (second picture), the ruffle retracts in a lever arm manner and pulls the beads toward the cell. (C and D) Sketch of the linear and the lever arm retraction respectively. (Scale bars: 5 μm.)

Fig. 2.

Nanometer-precise 3D tracking of linear and lever arm retraction. 2D projections (x–y and y–z plane) of 3D bead position histograms reveal whether a filopodial retraction is purely linear (A and B) or lever arm-like (E and F). The sampling rate of all of the 3D position measurements shown in this figure was 10 kHz. (A) The x–y (Upper) and y–z (Lower) histogram of linear retraction over a time interval of Δt = 1.9 s (the number of data points in each histogram is 1.9 s × 10 kHz = 19,000). The bin size of the position histograms is 2 nm in the x, y, and z direction. (B) The dynamics of the retraction process shown in A is visualized by displaying the y–z histogram for short time intervals: t = 0 to 0.4 s, 0.4 to 0.7 s, 0.7 to 0.9 s, etc. (in the background, the histogram shape is outlined for the whole time interval t = 0 to 1.9 s). (A and B) Part of the retraction occurs stepwise. The step sizes in this figures range from 30 to 42 nm. The counteracting optical force was <3 pN. (C) The z position vs. time traces of the retraction shown in A. The step transition times are between 30 ms and 120 ms. (D) Sketch of the linear, stepwise filopodial retraction. (E) The x–y (Upper) and y–z (Lower) histogram of lever arm retraction over a time interval of Δt = 3.0 s (the number of data points in each histogram is 3.0 s × 10 kHz = 30,000). The bin size of the position histograms is 2 nm in the x, y, and z direction. (F) The dynamics of the retraction process shown in E is visualized by displaying the y–z histogram for short time intervals: t = 0 to 0.5 s, 0.5 to 1.0 s, 1.0 to 1.5 s, etc. (in the background, the histogram is shown for the whole time interval t = 0 to 3.0 s). (G) Sketch of the lever arm retraction process.

Fig. 3.

Filopodial retraction step size. (A) Histogram (n = 70) of step sizes of linear filopodial retraction at counteracting forces ranging from 0 to 19 pN. The mean step size is s = 36 ± 13 nm (mean ± standard deviation). (B) Step size of linear filopodial retraction as a function of the counteracting force applied by the optical tweezers. In the force range from 0 pN to 3 pN, the step size was s = 40 ± 12 nm (n = 42); from 3 pN to 6 pN, the step size was s = 31 ± 12 nm (n = 15); and from 6 pN to 18 pN, the step size was s = 27 ± 11 nm (n = 12).

Fig. 4.

Myosin-Va RNA silencing: immunofluorescence and Western blot analysis. (A) Immunofluorescence analysis: myosin-Va antibody (DIL-2) labeling (red) and a DAPI staining (blue) of RAW cells. The siRNA 2 had a knockdown efficiency of ≈90%. (B) Western blot analysis: DIL-2 antibody against myosin-Va and the anti-α tubulin (Sigma) for the control (ctrl), the nonsilencing RNA (ns), and the silencing RNA (siRNA 1 and siRNA 2). The siRNA 2 against myosin-Va had a knockout-efficiency of ≈90%.

Fig. 5.

(A) Force–velocity curve for linear filopodial retraction: speed of linear filopodial retraction as a function of the counteracting force applied by the optical tweezers. Shown are the raw data points (crosses) and the pooled data points (squares) as well as the theoretical curve (blue line) according to Eq. 2. The data pooling was done over 3-pN force intervals. The open squares (which have no error bars) indicate data points where pooling was done over a single raw data point. (Inset) Two-state model for motor kinetics. Shown is the force-dependent free energy G of a two-state molecular motor system. State A and state B denote sequential motor positions along its track (reaction coordinate x). An external force F alters the free energy of the two states and thereby the transition rates. (B) Model for filopodial retraction. The data presented here are in agreement with the hypothesis that multiple myosin motors with a step size ≈36 nm are involved in filopodial retraction.