Swimming Euglena respond to confinement with a behavioral change enabling effective crawling

Abstract

Some euglenids, a family of aquatic unicellular organisms, can develop highly concerted, large amplitude peristaltic body deformations. This remarkable behavior has been known for centuries. Yet, its function remains controversial, and is even viewed as a functionless ancestral vestige. Here, by examining swimming Euglena gracilis in environments of controlled crowding and geometry, we show that this behavior is triggered by confinement. Under these conditions, it allows cells to switch from unviable flagellar swimming to a new and highly robust mode of fast crawling, which can deal with extreme geometric confinement and turn both frictional and hydraulic resistance into propulsive forces. To understand how a single cell can control such an adaptable and robust mode of locomotion, we developed a computational model of the motile apparatus of Euglena cells consisting of an active striated cell envelope. Our modeling shows that gait adaptability does not require specific mechanosensitive feedback but instead can be explained by the mechanical self-regulation of an elastic and extended motor system. Our study thus identifies a locomotory function and the operating principles of the adaptable peristaltic body deformation of Euglena cells.

Figure 1. Confinement triggers changes in cell behavior and metaboly.

(a) Euglena cells in dilute cultures (i) exhibited fast flagellar swimming without cell shape changes, at typical speeds of 68.2±1.13 μm/sec (SEM, n=50) or 1.31 ± 0.03 body lengths/sec (SEM, n=50). In crowded cultures (ii) cells displayed a variety of behaviors, including fast flagellar swimming (yellow arrow), cell rounding and spinning (red arrow), and large amplitude cyclic cell body deformations – metaboly (purple ellipse), Supplementary Movie S1. (b) When confined between glass plates, cells systematically developed metaboly. Observation using brightfield reflected light microscopy revealed the reconfigurations of the striated cell envelope (pellicle) concomitant with cell deformations in the plane of the glass plate (n=5 cells), Supplementary Movies S2 and S3. (c) Schematic representation of the pellicle, the strip separation w, and the local mechanism of active deformation of the pellicle, according to which the relative sliding between strips produces a shear strain γ along the direction of the strips. During the shape dynamics in (b) w remained nearly constant (559 ± 0.003 nm, SEM, n=100). (d) According to a mathematical model for the kinematics of the pellicle (Supplementary Note 1), the rate of change of γ with arc-length s along a strip (measured from the anterior end) is given by the curvature of the strip κ. By selecting strips in (b) emanating from the pole, where sliding is geometrically constrained, we could quantify the sliding displacement between adjacent strips wγ required to bend an initially straight strip (e), where the color-coding matches the strips highlighted in (b). (f) Cells swimming into tapered capillaries transitioned from fast flagellar swimming (i) to developing large amplitude shape excursions (ii) including rounding (iii) and the prototypical highly orchestrated peristaltic cell deformations of metaboly (iv), Supplementary Movie S4. This transition between (i) and (ii) occurred at a ratio between capillary and cell diameter of about 2.1 ± 0.05 (SEM, n=10).

Figure 2. Metaboly is an effective crawling mode of locomotion under confinement.

(a) At low confinement, the bulge traveling backwards along the cell body transiently contacted the capillary walls, producing a net forward motion (i). At higher confinement, cells adapted the gait, with a larger contact area of the bulge and smaller amplitude of cell deformations. However, the crawling mechanism remained the same (ii). (b) Kymographs of crawling Euglena gracilis under increasing confinement show the regularity of the gait, the backward bulge motion, and the forward cell motion. Crawling by metaboly was effective up to very large degrees of confinement (see the insets and Supplementary Movie S6). The normalized velocity expressed in cell body lengths per period was maximum at an intermediated capillary diameter (c), whereas the period of the gait was largely insensitive to confinement (d). The blue dashed lines in (c) and (d) are guides to the eye. The capillary diameter d_cap was normalized by the cell diameter d_cell as it was free-swimming with a fixed cigar shape in the wider part of the capillary (d_cell ≃ 9 μm). The error bars in (c) and (d) refer to the standard error of the mean and the size of samples is indicated. Here one sample is a complete period, and data was gathered from 16 cells.

Figure 3. Mechanism of locomotion during metaboly.

(a) (i) Canonical model of propulsion of non-adherent polarized animal cells under confinement: frictional forces induced by actin retrograde flow propel the cell forward against resistive hydraulic forces required to displace the water column in the capillary. (ii) In Euglena cells crawling by metaboly, the backward-moving pellicle bulge is analogous to actin retrograde flow in animal polarized cells. (b) Kinematics of the theoretical model for the power phase of metaboly. (i) Transformation of an idealized cylindrical pellicle by a uniform shear γ along the strips. (ii) By propagating a pellicle shear profile $\overline{\gamma }(s)$ along the cell body following $\gamma (s,t)=\overline{\gamma }(s-ct),$ we model a moving localized bulge, which with our sign convention, moves leftwards at speed c < 0 in the frame of the cell. (c) In the limit of infinite frictional coupling relative to hydraulic resistance (i), cell velocity is determined by the no-slip condition in the contact region. As indicated by the blue control volume, metaboly then results in net water pumping in the direction opposite to cell motion. The average fluid velocity vf is defined as the flow rate Q divided by the cross-sectional area of the capillary. In the limit of zero frictional coupling relative to hydraulic resistance (iii), cell velocity is determined by the no water pumping condition, cells are fastest, and the pellicle slides in the contact region, as indicated by the red arrows. In intermediate cases (ii), hydraulic forces (propulsive) and frictional forces (resistive) compete, there is some degree of sliding and pumping, and the cell velocity is intermediate. (d) Kymograph of an immobile cell next to another cell crawling by metaboly, Supplementary Movie 10. (e) Quantification of fluid flow around crawling cells tracking suspended beads. Error bars refer to the standard error of the mean and sample size is indicated (a sampling point is the instantaneous velocity of a bead between two frames, data from 2 cells and 21 bead trajectories). (f) Kymograph made from images intermittently focused at the capillary wall to visualize the pellicle and at the capillary axis to visualize cell shape. The trajectories of pellicle features (yellow curve) reveal sliding between the pellicle and the capillary wall in the contact region.

Figure 4. Computational modeling of crawling in confinement by metaboly.

(a) Model ingredients. (i) The pellicle is viewed as an elastic and extended motor system, modeled by an axisymmetric continuum surface with a field of material orientations converging towards two poles and accounting for the configuration of strips. The activation of this motor system in space (along the arc-length of strips, s) and time, t, is modulated to drive shape changes. Activation is modeled by the sliding velocity between adjacent strips in the absence of force, $v_{\text{motor}}^0(s,t).$ (ii) Time-periodic pattern of the activation $v_{\text{motor}}^0(s,t)$ during two gaits, in units of strip separation w per period T. (iii) The actual sliding velocity v_motor is modified by distributed forces along adjacent strips in the sliding direction, τ. We model the force-dependent velocity of the motor system with an affine relation characterized by the time and space dependent $v_{\text{motor}}^0$ and by a stall force τ_stall of fixed magnitude, see Supplementary Note 5 for a discussion. (iv) The distributed force acting on the motor system, τ(s, t), is determined by the mechanical interactions of the pellicle with its environment (cellular pressure; contact, frictional and hydraulic forces) and by the pellicle mechanics, which include bending elasticity and stretching elastic forces that penalize deviations between the actual pellicle shear-rate and that imposed by the motion of motors, v_motor(s,t)/w. (b) Selected snapshots during the gait at four degrees of confinement. (c) Kymographs obtained from simulations in the high fluid resistance (i) and the high wall friction (ii) limits at the same four degrees of confinement. The contact region is colored in orange and the lines represent trajectories of material particles on the pellicle surface, showing sliding in the hydraulic-dominated case and no sliding in the friction-dominated case. Induced flow rate, normalized by the maximum cell velocity times the capillary cross-sectional area, is represented by gray curves in the case of high friction; it is zero in the limit of high hydraulic resistance. (d) Normalized cell velocity in body lengths per period as a function of normalized capillary diameter predicted by the simulations.