Garno C et al. (2021), Building the cytokinetic contractile ring in an...

ECB-ART-50608

PLoS One 2021 Jan 01;1612:e0252845. doi: 10.1371/journal.pone.0252845.

Show Gene links Show Anatomy links

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

Garno C , Irons ZH , Gamache CM , McKim Q , Reyes G , Wu X , Shuster CB , Henson JH .

Abstract
The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

PubMed ID: 34962917
PMC ID: PMC8714119
Article link: PLoS One

Antibodies: anln Ab1 LOC590586 Ab2 phospho-LOC592760 Ab1 potekp1 Ab1 rhoa Ab2 septin2 Ab1

Article Images: [+] show captions

	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.
	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.
	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 Î¼m and magnifications of A-Q are equivalent. Bar in R = 10 Î¼m and magnification of R and S are equivalent. All cortices from S. purpuratus embryos.
	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 Î¼m; magnifications of A-L are equivalent. All cortices from S. purpuratus embryos.
	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 Î¼m x 3 Î¼m XY image on the top paired with a corresponding 10 Î¼m x 2 Î¼m 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.
	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 ROIâthe 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.
	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 Î¼m. All cortices from S. purpuratus embryos.
	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 Î¼m; magnifications of A-C are equivalent.
	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 Î¼m in A; magnifications of A-L are equivalent. L. pictus embryos in A-F, S. purpuratus embryos in G-L.

References [+] :

Argiros, Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos. 2012, Pubmed, Echinobase