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Fig 2. Anillin localizes to the cleavage furrow in first division embryos.In dividing S. purpuratus embryos co-labeled for microtubules (magenta) and DNA (white; blue in L) in order to determine mitotic progression, anillin (green) staining is not obvious in late anaphase (A-C) but begins concentrating in the early cleavage furrow starting at the initiation of telophase (D-F) and continues through the midbody stage prior to abscission in late telophase (G-O). In off axis images the anillin staining appears as an entire ring similar to other CR markers (J-L). In L the phase contrast image is superimposed on the fluorescence image for context. The magnifications of A-O are equivalent and A-I plus M-O are confocal images whereas J-L are widefield images.
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Fig 3. Septin2 and anillin colocalize with the CR marker P-MyoRLC in first division embryos.Both septin2 (A-L, green) and anillin (M-T, green) mirror P-MyoRLC (A-T, magenta) staining in dividing embryos and begin as collections of clusters (A-D, M-P) that progress to tight rings (E-H, Q-T), and end concentrated in the midbody (I-L). L. pictus embryos appear in confocal images in A-L, S. purpuratus embryos appear in widefield images in M-T, and the magnifications of A-T are equivalent.
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Fig 4. Widefield imaging of isolated cortices reveals that septin2 and anillin co-distribute with active myosin II and progress from clusters to more linearized arrays.Septin2, and active myosin II (P-MyoRLC) localize together in the CR regions of isolated cortices (A-H) and progress from regularly spaced clusters in early stages (A-D) to dense assemblages of more patchy and linear structures in late stages (E-H). Anillin displays a similar co-distribution with active myosin II, as well as the analogous evolution from clusters in early cortices (I-L) to denser and more filamentous arrays in mid-late cortices (M-Q). Lower magnification images of myosin II (MyoHC) staining in cortices isolated early in cytokinesis (R) shows the presence of punctate clusters in a majority of cortices containing CRs (percentages graphed in T), whereas cortices isolated mid-late in cytokinesis (S) have a majority of cortices with patchy/filamentous CR patterns (percentages graphed in T). Bar in A = 10 μm and magnifications of A-Q are equivalent. Bar in R = 10 μm and magnification of R and S are equivalent. All cortices from S. purpuratus embryos.
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Fig 5. Widefield imaging of isolated cortices demonstrates that septin2 and anillin colocalize with active myosin II, filamentous actin and RhoA/B/C in CRs.Septin2, active myosin II (P-MyoRLC) and F-actin staining all associate with clusters in early CRs (A-D), and with the denser, more linear arrays in late stage CRs (E-H). Anillin displays a similar association with active myosin II and F-actin in late stage CRs (I-L) and also codistributes with Rho A/B/C in the CR (M-P). Bar in A = 10 μm; magnifications of A-L are equivalent. All cortices from S. purpuratus embryos.
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Fig 6. 3D-SIM super-resolution imaging of clusters of myosin II, septin2 and anillin in early CR stage cortices.(A-F) Survey (A-C) and higher magnification views (D-F: enlarged white boxes in A-C) of isolated cortices from dividing embryos double labeled for P-MyoRLC (magenta in A-F) and either MyoHC (yellow in A, D), septin2 (green in B, E), or anillin (cyan in C, F). (G-L) The pairs of images that appear in G&J, H&K and I&L consist of a 10 μm x 3 μm XY image on the top paired with a corresponding 10 μm x 2 μm XZ image of the same clusters on the bottom. (M) In later stage CR regions of isolated cortices clusters become enlarged and appear to interact/coalesce with one another. Box and whisker plots (min/max with line at median) of small cluster spacing (N) and diameter (O) in early stage cortices stained by the three combinations of antibodies. Bar magnifications as indicated, with images in M equivalent in magnification to panel F. All cortices from S. purpuratus embryos.
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Fig 7. Analysis of early cluster organization using 3D SIM shows that septin2 and anillin tend to be central with myosin II on the periphery.(A-C) MyoHC (yellow) and P-MyoRLC (magenta) staining of early cytokinesis stage small clusters showing what appear to be mini-filaments arranged in chains (A) and rings (B, C). (D-G) Staining of P-MyoRLC (magenta) with either septin2 (green in D, E) or anillin (cyan in F, G) shows peripheral position of myosin II heads and the more central position of septin2 and anillin. (H-K) The central location of septin2 (green in H, I) and anillin (cyan in J, K) was confirmed by analyzing early small clusters with 2D line scans (H, J) and 3D surface plots (I, K) of relative staining intensities. Insets in H-K show images being analyzed and the line or area ROI–the images of clusters in I and K have been rotated to match the orientation of the 3D surface plots. (L) Box and whisker plots (min/max with line at median) of the percent of total early small clusters with centralized septin2 or anillin staining. Bar in A = 500 nm; magnifications of A-G are equivalent. All cortices from S. purpuratus embryos.
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Fig 8. 3D-SIM and STED super-resolution imaging of myosin II, septin2 and anillin in late stage CRs in isolated cortices.(A-D) MyoHC (yellow) and P-MyoRLC (magenta) staining of mature CR showing alignment of head-to-head minifilament chains (C, D). (E-L) Septin2 (green) and P-MyoRLC (magenta) labeling of a late stage CR region showing the network-like structure of septin2 filaments in close association with myosin II (H, L) using both 3D-SIM (E-H) and STED (I-L) imaging. (M-U) Anillin (cyan) and P-MyoRLC (magenta) staining of a mature CR indicates that anillin is more punctate in distribution and in close proximity to myosin II (M-P). The cortex in Q-V is highly contracted (Q shows a low magnification confocal view) and in this CR remnant the STED imaging of anillin appears similar to a network. White boxes in C, G, K, O, T correspond to the regions that appear at higher magnification in the insets labeled D, H, L, P, U. Bars = 10 μm. All cortices from S. purpuratus embryos.
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Fig 9. STED imaging of CR septin2 in isolated cortices shows a transformation from clusters to a filamentous network.(A-C) STED imaging of septin2 (green) and P-MyoRLC (magenta) in isolated S. purpuratus cortices shows elongate septin2 filaments in enlarging clusters in an early CR (A) versus the filamentous network structure of septin2 staining in later stage CRs (B, C). Insets in A-C show lower magnification confocal images of the cortices with the regions imaged by STED outlined by a white rectangle. Bar = 5 μm; magnifications of A-C are equivalent.
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Fig 10. Actin filaments are not required for the recruitment of myosin II, septin2 or anillin to early stage CR clusters.(A-C) Whole embryo (EMB) treated with LatB and stained for septin2 (green), P-MyoRLC (magenta), and DNA (blue) and viewed using a through focus projection of a confocal Z series. The embryo contains a circumferential band of clusters containing myosin II and septin2 and the DNA staining indicates that it has undergone nuclear division during mitosis but not cytokinesis. (D-F) Cortex (CTX) isolated from a LatB treated embryo shows that septin (green) and P-MyoRLC (magenta) localize to a concentrated stripe of clusters. (G-I) Whole embryo (EMB) treated with LatA and stained for anillin (cyan) and P-MyoRLC (magenta) shows they colocalize in a stripe in an embryo which has undergone karyokinesis (I, DNA in blue). (J-L) Cortex (CTX) isolated from a LatA-treated embryo demonstrating a band of anillin and P-MyoRLC clusters. Bar = 10 μm in A; magnifications of A-L are equivalent. L. pictus embryos in A-F, S. purpuratus embryos in G-L.
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