Membrane invaginations reveal cortical sites that pull on mitotic spindles in one-cell C. elegans embryos

Abstract

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Figure 1. Invaginations imaged by spinning disc confocal and SPIM.

A, Embryos expressed PH::GFP as a membrane marker were subject to the indicated treatment. Regions with invaginations, enclosed by rectangles, are enlarged below. Invaginations are marked by yellow arrows. Accumulation of the PH::GFP signal on the centrosomes occurs in all RNAi conditions and is indicated by red arrows. Scale bars are 5 μm. B, Selective plane illumination microscopy (SPIM) of a PH::GFP embryo treated with nmy-2(RNAi). Left: z-projection of 10 slices of the indicated region of the 3D image. The original image was reduced to 8 bit and median filtered. Three invaginations (yellow arrows) all point toward the centrosome (red arrow). Scale bar is 2 μm. Right: Level-set segmentation and surface reconstruction of the region showing the conical basis (black hole) of the invaginations (yellow arrows) from the cytoplasmic side of the cell membrane.

Figure 2. The number of invaginations depends on time following anaphase onset, location in the embryo and PAR-proteins.

A, Each dash represents an invagination (numbered in order of appearance on the ordinate axis), either formed on the posterior (blue) or anterior pole (red). The length of the dash indicates the duration of the invagination. The y-axis shows the index of the invagination, the x-axis gives the time in seconds. Onset of anaphase corresponds approximately to the start of the transverse oscillations shown underneath in blue. B, Number of invaginations in either the posterior (blue dot) or the anterior (red triangle) half of the embryo during anaphase increases with increasing duration of nmy-2 (RNAi) treatment. Grey lines pair the data coming from individual embryos. C, Comparison of invaginations formed in either the posterior (blue dot) or the anterior (red triangle) half of the embryo after 24 h of double nmy-2/mock(RNAi), 24 h of nmy-2 (RNAi) and 24 h of double par-2/nmy-2 (RNAi) respectively par-3/nmy-2 (RNAi); for corresponding values, see Fig. 3. Both in par-2/nmy-2 (RNAi) and par-3/nmy-2 (RNAi), no significant difference could be found between anterior and posterior count of invaginations.

Figure 3. Summary of invagination phenotypes for various genes involved in acto-myosin cortical organization and spindle pulling forces.

Invaginations were counted during the whole anaphase on the anterior and posterior pole under different RNAi conditions (mean ± Standard Deviation). Significant difference to control embryos for the anterior and posterior pole is indicated by black asterisks. Significant differences to nmy-2 (RNAi) or (nmy-2/mock (RNAi) in the case of double RNAi) for the posterior pole are indicated by open triangles. Proteins are classified by their proposed role, indicated by color shading. Grey for proteins involved in polarity, purple for force generation, yellow for kinesins, orange for actin and myosin regulators and turquoise actin crosslinkers.

Figure 4. Invaginations require microtubules.

Left panel shows a nmy-2 (RNAi) embryo expressing PH::GFP as a membrane marker treated with DMSO. An invagination is marked with a gray arrow. The right panel shows an nmy-2 (RNAi) embryo treated with nocodazole dissolved in DMSO: no invaginations were observed (see video S2).