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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01843

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Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity

Christine M. Field^1,, Margaret Coughlin¹, Steve Doberstein², Thomas Marty^3,* and William Sullivan⁴

¹ Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA
² Five Prime Therapeutics, South San Francisco CA 94080, USA
³ Howard Hughes Medical Institute, Developmental Genetics Program, Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
⁴ Department of Molecular, Cell and Developmental Biology, Sinsheimer Laboratory, University of Santa Cruz, Santa Cruz, CA 95064, USA

View larger version (38K): [in a new window]   Fig. 1. Genetic, sequence and western blot analysis of scraps (anillin) alleles. (A) An allelic series of scraps alleles from complementation data in Table S1. Genes with a stronger phenotype are on the right. (B) Schematic of Anillin domain structure; MY, Myosin II-binding region; ACT, F-actin binding region; AH, Anillin homology region; PH, pleckstrin homology domain, and (Septin binding) the region required for Anillin recruitment of septins to F-actin bundles. Positions of amino acid substitutions in anillin mutant alleles are shown below. (C) Sequence alignment near the N terminus of the Anillin PH domain. Sequences from Drosophila, human, Xenopus and C. elegans are shown with a predicted secondary structure below. The amino acid changes for the three strong scraps alleles are indicated above. Note that the V to S change at the beginning of the PH domain is present in all Schupach/Wieschaus alleles sequenced (red arrowhead). (D) Western blot analysis of maternal effect alleles. Embryo extracts from wild-type and three different maternal allelic combinations were probed with antibodies to Anillin and Actin.

View larger version (109K): [in a new window]   Fig. 2. Anillin and F-actin localization during cellularization. Indirect immunofluorescence of formaldehyde-fixed embryos using laser confocal imaging. (A,C) Wild-type, (B,D) anillinPQ/RS-derived embryos. Scale bar: 5 µm. (A) Wild-type embryo in slow phase, sectioned perpendicular to embryo surface. The teardrop-shaped furrow canals (white arrowheads) contain high levels of Anillin and F-actin. (a) Same embryo, sectioned parallel to the surface at the cellularization front. There is an almost hexagonal network of furrow canals, with Anillin and F-actin partially colocalized in bar-like structures. (B) anillinPQ/RS-derived embryo in slow phase, sectioned perpendicular to embryo surface. Furrow canals are malformed (white arrowhead), with lower Anillin levels than wild type. (b) Same embryo, sectioned parallel to the surface at the cellularization front. The network of furrow canals is malformed and partially disorganized, appearing fuzzy. (C) Wild-type embryo in late cellularization/early gastrulation, sectioned perpendicular to embryo surface. Ring-shaped Anillin and F-actin assemblies exist at the base of the newly formed cells (c,c'). The rings sit on top of stalks that connect the cells to the yolk mass (see Fig. S1 in the supplementary material). (D) anillinPQ/RS-derived embryo in late cellularization/early gastrulation, sectioned perpendicular to embryo surface. The ring-shaped Anillin and F-actin assemblies are missing (d,d'), and a disorganized F-actin network containing very little Anillin is present at the base of the cells.

View larger version (61K): [in a new window]   Fig. 3. Anillin and Myosin II localization during late cellularization. Indirect immunofluorescence of heat, followed by methanol, fixed embryos using laser confocal imaging. (A) Wild type; (B) anillinPQ/RS-derived embryos. Scale bar: 5 µm. (A) Wild-type embryo in late cellularization, sectioned perpendicular to embryo surface. The cellularization front is rich in colocalized Anillin and myosin II. Little Anillin is present in nuclei. (a-a'') Same embryo, sectioned parallel to the surface at the cellularization front. Contractile rings beneath each nucleus are rich in colocalized Anillin and Myosin II. (B) anillinPQ/RS-derived embryo in late cellularization, sectioned perpendicular to embryo surface. The cellularization front contains less Anillin. Much of the Anillin has moved into nuclei. Some of the nuclei are mis-positioned (white arrowhead). (b-b'') Same embryo, sectioned parallel to the surface at the cellularization front. Contractile rings are absent. Instead, Anillin and part of the Myosin II are present in abnormal bars. Some nuclei are visible in this plane of section (arrowheads), indicating mis-positioning relative to the embryo surface.

View larger version (63K): [in a new window]   Fig. 4. Anillin and Peanut (septin) localization during cellularization. Indirect immunofluorescence of cold methanol fixed embryos using laser confocal imaging. (A,C) Wild-type; (B,D) anillinPQ/RS-derived embryos. Scale bar: 5 µm. (A) Wild-type embryo in slow phase, sectioned perpendicular to embryo surface. The cellularization front is enriched in Anillin and Peanut. (a) Same embryo, sectioned parallel to the surface at the cellularization front, where Anillin and Peanut are almost colocalized. (B) anillinPQ/RS-derived embryo in slow phase, sectioned perpendicular to embryo surface. Although Anillin is still present at the cellularization front, Peanut is largely absent, instead it localizes to foci at the apical surface and throughout the cell (arrowhead). (b) Same embryo, sectioned parallel to the surface at the cellularization front. Most of the Peanut is missing, and the remaining protein is present in foci that do not colocalize with Anillin. (C) Wild type embryo in late cellularization/early gastrulation, sectioned perpendicular to embryo surface. Anillin and Peanut colocalize at the cellularization front. The apical and lateral plasma membrane contains Peanut but not Anillin (arrowhead). (c) Same embryo, sectioned parallel to the surface at the cellularization front. Anillin and Peanut colocalize in the rings. (D) anillinPQ/RS-derived embryo in late cellularization/early gastrulation, sectioned perpendicular to embryo surface. The cellularization front is disorganized, with most of the Anillin and Peanut missing. Within cells, Peanut is present in abnormal foci (white arrowhead) and nuclei are mis-positioned. (d) Same embryo, sectioned parallel to the surface at the cellularization front. Anillin and Peanut do not colocalize and contractile rings fail to form.

View larger version (113K): [in a new window]   Fig. 5. Time-lapse DIC analysis of cellularization front ingression. Embryo genotype as indicated. The top of each panel is the embryo surface. (A) In wild-type embryos, a distinct cellularization front (between arrows) can be seen passing through the nuclei. The entire cellularization process takes 70 minutes at 22°C and the membrane ingresses 35 µm from the embryo surface. (B) In mutant embryos, the cellularization front (between arrows) is less distinct. (C) Kymograph analysis of a wild-type embryo. A strip perpendicular to the embryo surface, the width of a nucleus, was cut out of time-lapse images as in A. Strips from each frame were pasted together to give a distance versus time plot (scale bars in F). The most obvious line in the kymograph, indicated by the white arrows, is the cellularization front. Its rate of movement is given by its angle: horizontal indicates static; vertical indicates fast moving. Ingression of the front occurs at three distinct rates, with breaks between them indicated by the white arrows. The black arrow indicates the base of the nucleus, that moves downwards as the nuclei elongate. (D) Second example of a kymograph of a wild-type embryo. (E) Kymograph of an anillinHP/RS-derived embryo (strong maternal alleles). No initiation phase is evident. As soon as the front can be tracked, it ingresses at a roughly constant and slow rate. A distinct transition to a faster rate can be observed (white arrow), but both slow and fast rates of ingression are slower that their wild-type counterparts. The black arrow indicates the base of the nucleus, which moves downwards as the nuclei elongate. (F) Kymograph of an anillinRV/RV-derived embryo (weak maternal allele). Ingression kinetics are similar to those in E.

View larger version (196K): [in a new window]   Fig. 6. Thin section transmission electron microscopy (TEM) analysis of cellularization. The boxed areas are presented at higher magnification in the panels on the right. (A-C) Wild-type embryo, fast phase. Note the almost triangular furrow canals (arrow in B) and the almost straight, closely apposed plasma membranes that connect the top of the furrow canal to the embryo surface (double arrow in C). (D-F) anillinHP/RS-derived embryo, slow phase. Furrow canals are lacking (D) or malformed (arrow in E). There are no paired plasma membranes at the top of the furrow canal in F. A line of vesicles is present in its place (double arrows). ER in F indicates an ER lamella. (G-I) anillinHP/RS-derived embryo in fast phase. The nucleus is mis-positioned (arrow in G). All plasma membranes between nuclei are vesiculated at this stage. Scale bar: 10 µm in A,D,G; 4 µm in B,E,H; 0.8 µm in C,F,I.

View larger version (136K): [in a new window]   Fig. 7. High magnification TEM of new plasma membranes. Left-hand panel shows a wild-type embryo. The paired plasma membranes are fairly straight, and a constant distance apart. (A-C) anillinHP/RS-derived, slow phase embryo (the same embryo shown in Fig. 6D-F). The panels show different positions in the same embryo, all between furrow canals and the surface. (A) The paired plasma membranes are almost normal, though the distance between them is variable. (B) The membrane are still almost continuous, but they are highly distorted and electron-dense material is present on the membrane. (C) The membrane is completely vesiculated, similar to the region shown in Fig. 6F. Scale bar: 150 nm.

View larger version (149K): [in a new window]   Fig. 8. TEM analysis of pole cells. Wild-type embryo in fast phase, showing a region near the posterior end of the embryo. PC, pole cell nucleus with characteristic round morphology with smooth nuclear envelope. CC, cellularizing cell nucleus. Stalks connecting the cells to the yolk mass (white arrows). (A) anillinHP/RS-derived embryo, late cellularization/early gastrulation. Cellularization is failing, but pole cell separation is almost normal. Boxed areas are shown at higher magnification in the insets. (B,b') Paired plasma membranes between two poles cells appear normal. (B,b'') Paired plasma membranes between two cellularized cells have completely vesiculated. anillinPE/PE derived embryo, late cellularization/early gastrulation. Cellularization is less perturbed than (C) in this weaker allele, but pole cell separation has failed completely. Boxed areas are shown at higher magnification in the insets. (c) No plasma membrane or vesicles are present between pole cell nuclei. (c') Partially vesiculated plasma membranes are present between the nuclei of cellularized cells. Scale bar: 2 µm in main panels, 0.5 µm in insets.

View larger version (91K): [in a new window]   Fig. 9. Immunofluorescence analysis of conventional cytokinesis. (A) Mitotic domain, wild-type embryo. Anillin and Peanut colocalize in cleavage furrows (*) and intracellular bridges (arrowheads). Intracellular bridges are long lasting, and were frequently observed in mitotic domains. (B) Mitotic domain, anillinPQ/RS-derived embryo. Mutant Anillin, but not Peanut, is localized to cleavage furrows (arrowhead) and to an apparently regressed cleavage furrow in a bi-nucleate cell (*). (C) Genotype as in B. Example of a rare intracellular bridge enriched in Anillin but not septins (arrowhead). Intracellular bridges were very rare, indicating that most attempts at cytokinesis fail. (D) Imaginal disc rudiment from 3rd instar larvae, genotype anillin7/anillinPQ stained for F-actin (red) and DNA (blue). Large binucleate cells (arrowheads) are present. Scale bar: 5 µm.

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