Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis

Abstract

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.

FIGURE 1:

NMII spot dynamics at the cortex and changes in localization of NMY-2::GFP in the cell during the assembly of the ring. (A) Spinning-disk confocal microscopy images of C. elegans one-cell embryos labeled with NMY-2::GFP (green) and the marker for actin filaments, LifeAct::mKate (magenta). Representative images of cells during the maintenance phase (t = −60 s), assembly of the ring (t = 0–60 s), and cytokinesis are shown (t = 120 s). Two enlarged sections are shown for each time point (zoom): one in the anterior and one in the equator where the ring will form (white dashed boxes). Scale bar: 10 µm; time is relative to the start of the assembly of the ring; A, anterior pole of the cell; P, posterior pole; inset, zoom. (B) Representative examples of NMII spots binding, dividing, and unbinding (scale bar, approximately 1 μm; time interval, 5 s. (D–G) Myosin density and intensity was plotted for the maintenance phase (blue), the start of ring assembly (dark green), the late ring assembly (light green), and the ring constriction (orange) for 10 spacial bins along the AP axis. The total NMII fluorescence intensity for the whole bin (D), the fluorescent intensity for segmented NMII spots (E), the number of NMII spots per unit of area of the cortex (F), and the spot intensity multiplied by the density (G) are plotted separately. Error bars: mean ± SD.

FIGURE 2:

NMII density and intensity increase at the posterior and decrease in the anterior during ring formation. The NMII spot density per μm² of the cortex is plotted over time for regions in the anterior and posterior (insets). NMII spot fluorescence intensity is plotted in the same way. Time is relative to the start of the assembly of the ring; t = –200-0 covers the end of maintenance phase; the vertical dashed line is the start of ring assembly; t = 0-200 covers ring assembly and constriction. The line and shaded area, mean of seven embryos ± SD; dotted line, average of 6 or less repeats.

FIGURE 3:

Superresolution imaging of NMII filaments in the cortex and show dynamics at the cortex and a sharp increase in the recruitment rate as the ring assembles. (A) Superresolution SI TIRF microscopy images of C. elegans one-cell labeled with NMY-2::GFP (green) and the marker for actin filaments, LifeAct::mKate (magenta). Representative images of cells during the maintenance phase (t = −60 s), assembly of the ring (t = 0-60 s) and during cytokinesis are shown (t = 120 s). Two enlarged sections are shown for each time point (insets): one in the anterior and one in the equator where the ring will form (white dashed boxes). Scale bar: 20 µm; time is relative to the start of the assembly of the ring. A, anterior pole of the cell; P, posterior pole. (B) Representative examples of NMII filaments binding to the cortex, dividing and unbinding (scale bar: 5 pixels or approximately 0.200 μm; time interval: 90 ms). (C) Color-coded tracks were plotted for individual tracked NMII filaments in the anterior and equator (insets). Each track starts at a position on the x-axis that corresponds to the time when a filament appeared at the cortex. The color of each track corresponds to the intensity of the filament (black, low intensity; green-yellow, higher intensities). These intensity profiles were then stacked on the y-axis in the order in which the corresponding filament appeared at the cortex (top first, bottom last) and a vertical red line shows the time when the ring starts to assemble. The top tracts represent the filaments that are in the field of view at the start of the movie. The white dashed lines are a guide to the eye for the rate of NMII filamnet recruitment to the cortex (arrival events per unit of time).

FIGURE 4:

Regulators of NMII activity affect the cortical dynamics and the assembly of the ring. (A) Scheme of the function of LET-502 and MEL-11. MEL-502 is activated by RHO-1 which in turn activates NMII by phosphorylating its RLC MLC-4 (purple circle). This converts inactive NMII (red) to the active conformation (green). MEL-11 deactivates NMII by removing the phosphate on MLC-4 and returning NMII to its inactive conformation. (B) microscopy images of cells during the maintenance phase subjected to RNAi against let-502 and mel-11. The localization of NMY-2::GFP (green) and LifeAct::mKate (magenta) is shown for the two RNAi conditions and wild type. Scale bar: 10 µm; yellow box, the size of the anterior cap. (C) PIV quantifying the flow of the cortex along the AP axis (V_x) in the posterior for the three conditions. Error bars: mean ± SD. (D) The NMII spot density per μm² of the cortex is plotted over time for regions in the anterior and posterior (insets) for let-502 RNAi (blue) and mel-11 RNAi (red) compared with wild type (green). NMII spot fluorescence intensity is plotted in the same way. Time is relative to the start of the assembly of the ring; line and shaded area, mean ± SD; dotted line, average for time points which were not covered by all repeats.

FIGURE 5:

Quantification NMY-2::GFP reveals changes in NMII concentration around the time the ring assembles that is affected by RNAi of let-502. (A) Spinning-disk microscopy images of NMY-2::GFP of the midplane of one-cell C. elegans embryos during the maintenance phase and during assembly of the cytokinetic ring. Dashed white boxes show where the NMY-2::GFP signal is excluded from the pronucleus (maintenance phase) and enriched in the spindle (assembly of the ring). The output of the segmentation of cytoplasmic is show which robustly identifies cytoplasmic NMII spots (arrowheads) and can distinguish them from cortical NMII in the plane perpendicular to the imaging plane (arrows). Scale bar: 5 µm; time is relative to the start of the assembly of the ring as judged for the cortical plane; A, anterior pole of the cell; P, posterior pole. (B) The average pixel intensity for cytoplasmic NMY-2::GFP at midplane plotted over time for wild type (green), let-502 RNAi (blue), and mel-11 RNAi (red). The data are for a rectangular ROI covering the whole AP axis but excluding the cell periphery (inset). Intensity values are normalized such that the intensity during the maintenance phase (t = −150) is 1, meaning that the data represent the relative change in NMY-2::GFP intensity over time. (C) The NMII spot density per unit volume of the whole cytoplasm plotted over time calculated from the spot mask. Dashed line, 0.01 counts/µm³.

FIGURE 6:

The gradient and NMII concentrations in the cytoplasm are affected by RNAi of let-502 and mel-11. (A) Nonnormalized pixel intensity values for NMY-2::GFP in the cytoplasm for 18 spatial bins along the AP axis (inset) for the time point 60 s before the onset of ring assembly. The values are for the total pixel intensities including the diffuse signal and the spots (inset). The mean and SD of the data are plotted for wild type in green (solid line, mean; shaded area, ± SD), let-502 RNAi in blue and mel-11 RNAi in red. Each dataset was fitted with a fourth-order plolynomial (dashed line), and the gradient was measured by calculating the gradient at the point of inflection (open circle). (B) Bar chart of the NMY-2::GFP intensity at the site of the future division plane (corresponding to bin 10 in panel A).

FIGURE 7:

Scheme of cortical and cytoplasmic actomyosin in the one-cell C. elegans embryo. The distribution and assembly of actomyosin in the cortex and cytoplasm are represented during the maintenance phase and during ring assembly. Actin filaments are represented by pink rods and NMII are green sticks. The cytoplasmic concentration of NMII is represented by varying intensities of green. The arrows represent a dynamic exchange of NMII filament binding to and unbinding from the cortex. Cytoplasmic NMII becomes deactivated resulting in NMII filaments disassembling into free NMII motors (curly green sticks) which are then free to diffuse. We propose that MEL-11 promotes the disassembly of NMII at the anterior cap during the maintenance phase. This enriches NMII in the posterior half of the cytoplasm. In the posterior, the cortical concentration is low because of the movement of NMII to the anterior during flow phase, resulting in a lower off rate of NMII detaching from the cortex there. These two factors establish a cytoplasmic gradient of NMII that is high in the anterior and low in the posterior. Free motors in the cytoplasm can diffuse down the concentration gradient (zig-zag arrow) to the site of the future division plane. During ring assembly, LET-502 actives NMII, recruiting it to the ring thereby depleting motors from the cytoplasmic pool. Free motors assemble into filaments that bind to the cortex or are added to filaments that are already bound.