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First published online 21 January 2004
doi: 10.1242/dev.00989

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src64 and tec29 are required for microfilament contraction during Drosophila cellularization

Howard Hughes Medical Institute, Molecular Biology Department, Washington Road, Princeton University, Princeton, NJ 08544, USA

View larger version (50K): [in a new window]   Fig. 1. Src64, Tec29 and anillin localization in wild-type and src64 mutants during cellularization. Cross-sections of a wild-type (A) and a src6417 mutant (B) embryo, and sagittal sections of wild-type (C,E,G) and src6417 mutant (D,F,H) embryos during cellularization. Embryos were stained with antibodies to Src64 (A,B), Tec29 (C,D), phosphotyrosine (E,F) and anillin (G,H). (A) Src64 protein localizes predominantly to the cellularization front in wild-type embryos but is absent in src6417 mutant embryos (B). (C,D) Tec29 localizes strongly to the cellularization front and less strongly to the apico-lateral membrane in both wild-type and src64 embryos. (E,F) Phosphotyrosine-containing proteins localize to the cellularization front in both wild-type embryos and src64 embryos. (G,H) Anillin localizes to the cellularization front in both wild-type and src64 embryos. Scale bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 2. src64 is required for microfilament ring contraction during cellularization. Cross-sections (A,B,E,F) and projections of confocal sections of the cellularization front (C,D,G,H) of wild-type (A,C,E,G) and src6417 mutant (B,D,F,H) embryos, before (A-D) and after (E-H) the cellularization front has passed the bases of the nuclei. Embryos were stained with antibodies to myosin (A-H) and Armadillo (E,F). The early cellularization front, shown by myosin localization, is of uniform depth along the circumference of wild-type embryos (A), but is of non-uniform depth in src64 mutant embryos (B). The newly formed microfilament rings are round during early cellularization in wild-type embryos (C), but are less rounded in src64 mutant embryos (D). The late cellularization front is of uniform depth along the circumference of wild-type embryos (E) and the furrow canals are expanded into a flask-like shape, whereas in src64 mutant embryos, the late cellularization front is also of uniform depth but the furrow canals are unexpanded (F). The microfilament rings of wild-type embryos during late cellularization are round and constricted (G), whereas the microfilament rings of src64 mutant embryos are less rounded and are not constricted (H), similar to the microfilament rings of src64 mutant embryos during early cellularization (D). Scale bar: 10 µm.

View larger version (11K): [in a new window]   Fig. 3. src64 is not required for membrane invagination during cellularization. For both wild-type and src64 embryos, the progress of membrane invagination was measured from the cell apices to the cellularization front and plotted at five minute intervals starting at the beginning of cycle 14 (time=0 minutes) at 25°C. Maximum cellularization depth is obtained just before gastrulation begins in the interval between 55 minutes and 60 minutes. During early cellularization, the s.e.m. values are between 0.1 µm and 0.6 µm, whereas during late cellularization, the s.e.m. values are between 0.5 µm and 1.0 µm.

View larger version (111K): [in a new window]   Fig. 4. tec29 is required for microfilament ring contraction during cellularization. (A,B) Projections of confocal sections of the cellularization front after it has passed the nuclear bases of a wild-type (A) and a tec29k00206 germline clone (B) embryo. Embryos were stained with antibody to myosin. The microfilament rings of wild-type embryos during late cellularization are round and constricted (A), whereas the microfilament rings of tec29 germline clone mutant embryos are less rounded and are not constricted (B). Scale bar: 10 µm.

View larger version (116K): [in a new window]   Fig. 5. scraps (anillin) is required for the formation of microfilament rings during cellularization. (A-D) Sagittal sections (A,C) and projections of confocal sections of the cellularization front (B,D) of a scrapsRS/scrapsPQ mutant embryo shortly before (A,B) and after (C,D) the cellularization front has passed the nuclear bases. Embryos were stained with antibody to myosin. (A) The furrow canals of scraps mutant embryos during early cellularization are only slightly abnormal. (B) Microfilament rings are not present in scraps mutant embryos during early cellularization and the basal lumens are less rounded than in wild-type embryos. Myosin is found in aggregates scattered along the cellularization front (compare with Fig. 2C). (C) The furrow canals of scraps mutant embryos during late cellularization are collapsed and lack a flask-like morphology. (D) Microfilament rings are not present in scraps mutant embryos during late cellularization; the basal lumens are angular and are less rounded than those during early cellularization in scraps mutant embryos (compare with B). Myosin is found in aggregates scattered along the cellularization front (compare with Fig. 2G). Scale bar: 10 µm.

View larger version (76K): [in a new window]   Fig. 6. Bottleneck protein co-localizes to the cellularization front with myosin. Cross-sections (A,C,E) and projections of confocal sections of the cellularization front (B,D,F) before the cellularization front has passed the nuclear bases of wild-type embryos. Embryos were stained with antibodies to Bnk and myosin, and images have been arranged to show Bnk protein (A,B), Myosin (C,D), and merged images of both Bnk and myosin (E,F). (A,B) Bnk is expressed along the entire furrow canal and in microfilament rings during early cellularization. (C,D) Myosin is expressed basal-laterally in the furrow canal and in microfilament rings. (E,F) Bnk and myosin localization overlaps in microfilament rings and overlaps basal-laterally in the furrow canal, but Bnk localization extends farther apically. Scale bar: 10 µm.

View larger version (148K): [in a new window]   Fig. 7. Phenotypes of bnk and scraps are co-expressed. Projections of confocal sections of the cellularization front (A-C) and sagittal sections (D-F) of the same bnk mutant (A,D), scrapsRS/scrapsPQ mutant (B,E) and scrapsRS/scrapsPQ; bnk double-mutant (C,F) embryos. Embryos were stained with antibody to myosin (A-F) and with Hoechst dye (D-F). (A) Microfilament rings are hypercontracted in bnk mutant embryos; some nuclei are constricted into dumbbell shapes by the hyperconstricted microfilament rings and carried out of the periphery by the cellularization front in bnk embryos (D). (B) scraps mutant embryos showing the absence of microfilament rings, angular basal lumens and a normal nuclear morphology (E). (C) Microfilament rings are not formed in scraps; bnk mutant embryos, but the cellularization front still exhibits increased contraction and large gaps in the microfilament network. (F) Despite the absence of microfilament rings in scraps; bnk mutant embryos, some nuclei are constricted into dumbbell shapes by the contracted microfilaments and carried out of the periphery. Scale bar: 10 µm.

View larger version (105K): [in a new window]   Fig. 8. src64 activity is required for bnk hypercontraction. Projections of confocal sections of the cellularization front (A-C) after it has passed the nuclear bases of a bnk mutant (A), a src64 mutant (B) and a src64 bnk double-mutant (C) embryo. Embryos were stained with antibody to myosin (A-C). (A) bnk mutant embryo showing hypercontracted microfilament ring phenotype. (B) src64 mutant embryo showing non-contracted microfilament ring phenotype. (C) src64 bnk double-mutant embryo showing a non-contracted microfilament ring phenotype similar to that of src64 mutant embryos (B). Scale bar: 10 µm.

View larger version (23K): [in a new window]   Fig. 9. Model for src64-dependent and src64-independent forces acting during cellularization. (A) Epistasis pathway for microfilament contraction during cellularization. src64 is epistatic to bnk in the pathway controlling microfilament ring contraction. tec29 is speculatively placed in the pathway with src64 as Tec kinases are generally activated by Src kinases, although this has not been experimentally confirmed for Drosophila cellularization. scraps (anillin) has been placed in a parallel pathway to bnk and src64 because it plays an important, but indirect, role in microfilament ring contraction by stabilizing microfilament rings. In the absence of anillin, microfilament rings are not formed, but the disorganized microfilaments still have the ability to contract in the absence of bnk activity. peanut (pnut) also acts in this process. (B) Mechanical model for the interaction of Bnk and Src64 protein during early cellularization. See Discussion for details. (C) Mechanical model for the interaction of Bnk and Src64 protein during late cellularization. See Discussion for details.

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