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FIGURE 1:. The CRs in isolated cortices contain characteristic protein constituents. Wide-field imaging of immunofluorescently labeled cleavage cortices reveals that the CR regions contain the expected protein constituents, namely actin (B, J, N; magenta in D, L, P), myosin II heavy chain (Myo II HC in C, F; green in D, H), Ser-19–phosphorylated myosin II regulatory light chain (P-Myo II RLC in G; red in H), septin 2 (K; yellow in L), and RhoA (O; blue in P). Phase contrast images (A, E, I, M) show the indented/butterfly-like appearance of cortices isolated during cytokinesis. Bars, 10 µm (A, M); magnification equivalent in A–L.
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FIGURE 2:. SIM imaging indicates that CR region myosin II filaments undergo a transition from clusters to linear arrays during cytokinesis. The 3D SIM through-focus projections (120 nm/slice over ∼600 nm of Z) of early-stage cortical CR regions stained for myosin II HC (A; green in C) and P-Myo II RLC (B; red in C) reveal the presence of a broad band of clusters of myosin II filaments. Higher magnification (inset in C) reveals that clusters often appear as rings with linear extensions. NNI analysis of cluster spacing in early cortices from three experiments indicates that they are distributed in a uniform/regular manner (J). Mid-stage cleavage cortices (D–F) show the presence of myosin II clusters in larger patches associated with faint linear elements (arrows). At higher magnification (inset in F). the myosin II filaments appeared to be arranged into networks. CRs in late-cleavage-stage cortices (G–I) are characterized by striking linear arrays (arrows) of aligned myosin II filaments (inset in I) in a narrower band. Measurement of the width of the overall CR region myosin II staining over time in three separate experiments (K) indicates a progressive narrowing coincident with progression from early to late-cleavage-stage cortices. Bar, 10 µm.
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FIGURE 3:. SIM imaging of myosin II filament orientation in the CR emphasizes a change from clusters to linear arrays and indicates an association with actin filaments. The 3D SIM through-focus projections (120 nm/slice over ∼600 nm of Z) of early-stage (A), mid-stage (B), and late-stage (C) cleavage cortices stained with MyoII-HC (green) and P-Myo II RLC (red) antibodies. Early cortices (A) contain ring-shaped clusters of myosin II filaments, whereas in mid-stage cortices (B), the clusters appear to have linear extensions and have interconnected into networks. In late-stage cortices, the CR region contains an apparent linear arrangement of myosin II filaments that at high magnification (D) resolves into an alternating pattern of green and red staining, suggesting that myosin II filaments are in longitudinal chains of head-to-head–oriented filaments. Treatment with the actin-disrupting drug latrunculin A allows myosin II to assemble in the CR region (F, G), but the myosin arrangement does not reach the linearized pattern seen in the cortices isolated from equivalent-time-point untreated embryos (E). Triple labeling of a cortical CR with fluorescent phalloidin (J, blue in K), Myo II HC (H, green in K), and P-Myo II RLC (I, magenta in K) reveals that actin filaments concentrate in the CR in concert with myosin II filaments. Bars, 2 μm (A–C), 200 nm (D), 5 μm (E–K).
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FIGURE 4:. TEM of non–detergent-extracted cortices reveals submembranous actin-like filaments and membranous organelles in the CR region. (A) Low-magnification view of an indented cortex with microvilli on the edge and the surface covered with membranous vesicles and ER remnants. The red box appears at higher magnification in B, and the blue box appears at higher magnification in C. (B) Microvilli and numerous vesicles are evident in this image with some limited views of the cytoplasmic face of the plasma membrane. (C) Examination of an area with fewer vesicles at higher magnification reveals an underlying mat of actin-like filaments with an overall parallel alignment that we interpret as the presumptive CR. The yellow box appears at higher magnification in D. (D) High-magnification view of the submembranous actin-like filaments in putative contractile ring, showing that the majority are aligned into uniform bundles, although there are filaments running in other directions. Note the vesicles, remnants of the ER (center), and a clathrin-coated vesicle (top, center). (E, F) In regions not associated with the CR, the cortex still contains many submembranous actin-like filaments (E, low magnification; F, high magnification), although they assume a more isotropic arrangement. Scale bars as indicated.
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FIGURE 5:. TEM of detergent-extracted cortices shows that the CR consists of a closely packed array of actin-like filaments. A dense mat of filaments is apparent in the CR region of buttery shaped (A) and detergent-extracted cleavage cortices seen at low (A, D), medium (B, from red box in A; E, from green box in D), and high (C, from blue box in B; F, from yellow box in D) magnification. The CR region consists of tightly packed, elongate, largely unbranched, and aligned actin-like filaments, with other filament types and globular structures (H) intercalated into the structure. Note that the majority of CR filaments appear aligned parallel to the axis of constriction of the cleavage furrow and that clathrin-coated vesicles and membranous remnants are also present. In areas of cortices not associated with the CR, isotropic arrangements of actin-like filaments predominate (G). Comparison of the actin-like filament assemblage in sea urchin CR (H) with a mammalian LLC-PK1 cell stress fiber (I) emphasizes the similarity between these two structures. Scale bars as indicated.
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FIGURE 6:. TEM of myosin S1–labeled cortices allows for the definitive identification of CR actin filaments. Low- (A, C), and high- (B, D, inset in D) magnification images of the CR regions (demarcated by white arrows in A, C) of two indented cleavage cortices shows the characteristic twisted-rope appearance of myosin S1–decorated actin filaments. Within the CR regions, these actin filaments appear elongate, unbranched, and densely packed, with the majority aligned parallel to the cleavage plane. The actin filament orientation appears mixed, as demonstrated by the red arrows in D and the inset in D. (G) Quantitative analysis of F-actin anisotropy in five different cortices measured within (E) and outside (F) of the CR region. Note that F-actin anisotropy is statistically significantly higher (p < 0.0001) within the CR region. (H) The roughly similar percentages of plus and minus end–oriented filaments that were aligned with the CR axis, with a smaller number of off-axis filaments. Scale bars as indicated.
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FIGURE 7:. TEM of detergent-extracted, gelsolin-treated cortices reveals tightly packed bundles of myosin II filaments within the CR. Linear arrays of myosin II filaments are evident in detergent-extracted and gelsolin-treated cleavage cortices imaged at low (A, E), medium (B, from red box in A; F, from purple box in E), and high (C, from blue box in B; D, from orange box in B; G, from cyan box in F) magnification. The myosin II filaments are recognized by their characteristic appearance of globular heads and smooth rod/tail regions, their 200–300 nm length (K), and their alignment into head-to-head–associated longitudinal chains. In D and G, a subset of myosin II filament heads have been colored red and tails/rods green, and the gold-bordered insets at the top right show similarly colored isolated myosin II filaments from coelomocytes for comparison. The bottom right inset in D is a high-magnification view of an array of myosin II filaments from a similarly colored cortical CR. The myosin II filaments within the CR region tend to align parallel to the long axis of the CR/cleavage plane (axis indicated by dashed white line in C and G). Myosin II filament organization in the CRs is very similar in appearance to the array of myosin II filaments seen in TEM of gelsolin-treated stress fibers from cultured mammalian LLC-PK1 cells (H; the large filaments in the background are intermediate filaments). In some cortices, myosin II filaments are arrayed in a more isotropic network organization (I, low magnification; J, high magnification from green box in I), which are reminiscent of the patches of myosin II filament networks seen in SIM images of mid-stage cortices (Figures 2 and 3). Scale bars as indicated.
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