Dictyostelium myosin II mechanochemistry promotes active behavior of the cortex on long time scales

Abstract

Cell cortices rearrange dynamically to complete cytokinesis, crawlin response to chemoattractant, build tissues, and make neuronal connections. Highly enriched in the cell cortex, actin, myosin II, and actin crosslinkers facilitate cortical movements. Because cortical behavior is the consequence of nanoscale biochemical events, it is essential to probe the cortex at this level. Here, we use high-resolution laser-based particle tracking to examine how myosin II mechanochemistry and dynacortin-mediated actin crosslinking control cortex dynamics in Dictyostelium. Consistent with its low duty ratio, myosin II does not directly drive active bead motility. Instead, myosin II and dynacortin antagonistically regulate other active processes in the living cortex.

Fig. 1.

LPT system for measuring cortical mechanics of Dictyostelium discoideum. (A) Myosin-II ATP hydrolysis cycle. The rate-limiting step for motility is k_ADP-release, which is 420 s⁻¹, yielding a strongly bound-state time τ_s of 2.4 ms (5). (B) The LPT system uses a low-power laser that focuses on a 0.7 μm-carboxylated polystyrene bead attached to the surface of a Dictyostelium cell. Bead motions deflect the laser beam, which is relayed to a quadrant photodiode detector. (C) Differential interference contrast microscopy (DIC) and fluorescence imaging of coronin-GFP (46), GFP-dynacortin (28), and GFP-myosin II (44) shows that the bead (Left, arrows) does not recruit coronin, dynacortin, or myosin II. Surface (Center) and middle (Right) sections of deconvolved images are shown for comparison. (Scale bar, 10 μm.) (D) Total internal reflection fluorescence microscopy demonstrates that myosin II thick filaments are assembled in the cortex. (E) Western analysis using anti-myosin II monoclonal and anti-dynacortin polyclonal antibodies of cells expressing various myosin II and dynacortin constructs.

Fig. 2.

Beads on wt cortices show time scale-dependent subdiffusive and superdiffusive behaviors. (A) An example of a bead trace that showed largely diffusive behavior directed along a linear path over a 20-s interval. (B) Bead path in A after linear detrending to remove long-distance motility that probably results from cell crawling. (C) Plot of nondetrended MSDs vs. correlation time τ for 10 beads, showing SSP behavior for time scales shorter than ≈1 s. (D) Average MSD plots for nondetrended and detrended trajectories for the class of beads that show SSP behavior. (E) Distribution of power laws calculated from the detrended data at τ = 1 s by using a three-quarter-decade interval (from 0.4 to 2.4 s) for this class of bead behaviors. (F) An example of a bead path that showed SSA behavior. (G) Bead path in F after linear detrending. (H) Plot of nondetrended MSDs vs. τ for 10 beads showing SSA behavior on time scales more than ≈200 ms. (I) Average MSD plots for nondetrended and detrended trajectories for the class of beads that show largely superdiffusive behavior on time scales >200 ms. (J) Distribution of power laws calculated from the detrended data at τ = 1 s by using a three-quarter-decade interval (from 0.4 to 2.4 s) for this class of beads.

Fig. 3.

All genotypes show similar types of SSA and SSP bead behaviors but differ in the frequency of each type of behavior. (A–G) Histograms of distributions of power law, γ, at τ = 1 s for detrended data. Any bead that had γ > 1 at any point was shaded dark gray, regardless of its γ at precisely τ = 1 s. (A) wt control, n = 72. (B) myoII:GFPmyoII, n = 41. (C) wt:myoIIhp, n = 44. (D) myoII control, n = 106. (E) wt:dynhp, n = 35. (F) myoII:dynhp, n = 96. (G) myoII:S456L, n = 49. (H) Frequency histogram representing the fraction of beads in each class for each genotype.

Fig. 4.

Spectra of MSD magnitudes for two bead classes demonstrate that dynacortin has the largest impact on the SSP class. (A) wt control vs. wt:myoIIhp. (B) myoII:GFPmyoII vs. myoII control. (C) myoII control vs. myoII:S456L. (D) wt control vs. wt dynhp. (E) myoII control vs. myoII:dynhp. Error bars are SEMs. The n values are as shown in the Fig. 3 legend.

Fig. 5.

Summary of myosin II and dynacortin control of active cortical processes. Frequency bars depict the fraction of particles in the SSA vs. SSP class for each protein and is drawn from data in Fig. 3H. Color shading reflects the magnitude of the MSDs. Blue bars represent the SSA class, and red bars represent the SSP class.