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Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

Sisson JC, Field C, Ventura R, Royou A, Sullivan W - J. Cell Biol. (2000)

Bottom Line: Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated.Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190.We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, Sinsheimer Labs, University of California at Santa Cruz, Santa Cruz, California 95064, USA. sisson@darwin.ucsc.edu

ABSTRACT
Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

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Anti–Lva IgG antibody inhibits furrow progression. All panels are from scanning confocal movies of Myo-GFP embryos. (A, left) Anti–GST antibody-injected embryo undergoing normal furrowing. The red arrowheads indicate an injection artifact that the embryo repairs. (Right) Anti–Lva IgG antibody (3.8 mg/ml)-injected embryo undergoing disrupted furrowing (vertical arrows). A surface dimple forms near the site of injection where furrowing is most impaired (horizontal arrow). The Myo-GFP puncta observed near the site of the anti–Lva antibody injection (white arrowheads) and the smaller Myo-GFP puncta observed normally in both embryos do not colocalize with anti–Lva/Cy5 antibody marked Golgi (data not shown). Time (minutes) is relative to the start of cycle 14. The dorsal, injected surface of each embryo is shown. All views are sagittal. Bar, 10 μm. (B) Sagittal views of two cellularizing embryos that were injected with either anti–GST (left) or anti–Lva (right) IgG antibodies, fixed, and stained with anti–Golgi p120 antibody. Brackets indicate the position of nuclei (apical is up). Bar, 5 μm.
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Figure 6: Anti–Lva IgG antibody inhibits furrow progression. All panels are from scanning confocal movies of Myo-GFP embryos. (A, left) Anti–GST antibody-injected embryo undergoing normal furrowing. The red arrowheads indicate an injection artifact that the embryo repairs. (Right) Anti–Lva IgG antibody (3.8 mg/ml)-injected embryo undergoing disrupted furrowing (vertical arrows). A surface dimple forms near the site of injection where furrowing is most impaired (horizontal arrow). The Myo-GFP puncta observed near the site of the anti–Lva antibody injection (white arrowheads) and the smaller Myo-GFP puncta observed normally in both embryos do not colocalize with anti–Lva/Cy5 antibody marked Golgi (data not shown). Time (minutes) is relative to the start of cycle 14. The dorsal, injected surface of each embryo is shown. All views are sagittal. Bar, 10 μm. (B) Sagittal views of two cellularizing embryos that were injected with either anti–GST (left) or anti–Lva (right) IgG antibodies, fixed, and stained with anti–Golgi p120 antibody. Brackets indicate the position of nuclei (apical is up). Bar, 5 μm.

Mentions: Because no mutations are known to exist in the lva gene, we injected a concentrated preparation (3.8 mg/ml) of the anti–Lva antibody into embryos at the start of cycle 14 to block Lva function, and recorded the effects on Golgi bodies and furrow progression. To visualize the furrow front in these experiments, we used embryos bearing a spaghetti squash (sqh)–green fluorescent protein (gfp) transgene (Royou et al. 1999). sqh encodes the regulatory light chain of Myosin II, which normally accumulates at the furrow front (Karess et al. 1991). Likewise, in sqh-gfp embryos, functional myosin II-GFP (Myo-GFP) reveals the furrow front (Royou et al. 1999). The rate and extent of furrow progression is normal in control embryos injected with anti–GST antibody (Fig. 6 A). However, injections of the anti–Lva antibody cause severe furrowing defects (Fig. 6 A). At ∼50 min into cycle 14, furrow progression is clearly inhibited near the site of injection, where the antibody is most concentrated (Fig. 6 A). Further from the injection site, furrowing occurs at a reduced rate. Approximately 60 min into cellularization, the embryo's surface near the site of injection begins to dimple, becoming pronounced 10 min later (Fig. 6 A). This effect is presumably due to the pulling force exerted by the contractile apparatus on the surface of the embryo where furrowing has failed. Dimpling was never observed in control embryos. To directly examine the effects of the anti–GST and anti–Lva antibodies on Golgi, some injected embryos were fixed and analyzed by immunofluorescence using the anti–p120 cis-Golgi antibody. In control anti–GST antibody injected embryos, a normal distribution of p120 is observed (Fig. 6 B, left). However, in embryos injected with the concentrated anti–Lva antibody, the normal punctate distribution of p120 is not observed and instead p120 is found throughout the apical cytoplasm (Fig. 6 B, right). When the concentrated anti–Lva antibody is Cy5 tagged, the Lva protein can be seen to disperse within minutes of the injection, and the residual Lva protein left associated with some Golgi show them broken down and sessile (data not shown).


Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

Sisson JC, Field C, Ventura R, Royou A, Sullivan W - J. Cell Biol. (2000)

Anti–Lva IgG antibody inhibits furrow progression. All panels are from scanning confocal movies of Myo-GFP embryos. (A, left) Anti–GST antibody-injected embryo undergoing normal furrowing. The red arrowheads indicate an injection artifact that the embryo repairs. (Right) Anti–Lva IgG antibody (3.8 mg/ml)-injected embryo undergoing disrupted furrowing (vertical arrows). A surface dimple forms near the site of injection where furrowing is most impaired (horizontal arrow). The Myo-GFP puncta observed near the site of the anti–Lva antibody injection (white arrowheads) and the smaller Myo-GFP puncta observed normally in both embryos do not colocalize with anti–Lva/Cy5 antibody marked Golgi (data not shown). Time (minutes) is relative to the start of cycle 14. The dorsal, injected surface of each embryo is shown. All views are sagittal. Bar, 10 μm. (B) Sagittal views of two cellularizing embryos that were injected with either anti–GST (left) or anti–Lva (right) IgG antibodies, fixed, and stained with anti–Golgi p120 antibody. Brackets indicate the position of nuclei (apical is up). Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 6: Anti–Lva IgG antibody inhibits furrow progression. All panels are from scanning confocal movies of Myo-GFP embryos. (A, left) Anti–GST antibody-injected embryo undergoing normal furrowing. The red arrowheads indicate an injection artifact that the embryo repairs. (Right) Anti–Lva IgG antibody (3.8 mg/ml)-injected embryo undergoing disrupted furrowing (vertical arrows). A surface dimple forms near the site of injection where furrowing is most impaired (horizontal arrow). The Myo-GFP puncta observed near the site of the anti–Lva antibody injection (white arrowheads) and the smaller Myo-GFP puncta observed normally in both embryos do not colocalize with anti–Lva/Cy5 antibody marked Golgi (data not shown). Time (minutes) is relative to the start of cycle 14. The dorsal, injected surface of each embryo is shown. All views are sagittal. Bar, 10 μm. (B) Sagittal views of two cellularizing embryos that were injected with either anti–GST (left) or anti–Lva (right) IgG antibodies, fixed, and stained with anti–Golgi p120 antibody. Brackets indicate the position of nuclei (apical is up). Bar, 5 μm.
Mentions: Because no mutations are known to exist in the lva gene, we injected a concentrated preparation (3.8 mg/ml) of the anti–Lva antibody into embryos at the start of cycle 14 to block Lva function, and recorded the effects on Golgi bodies and furrow progression. To visualize the furrow front in these experiments, we used embryos bearing a spaghetti squash (sqh)–green fluorescent protein (gfp) transgene (Royou et al. 1999). sqh encodes the regulatory light chain of Myosin II, which normally accumulates at the furrow front (Karess et al. 1991). Likewise, in sqh-gfp embryos, functional myosin II-GFP (Myo-GFP) reveals the furrow front (Royou et al. 1999). The rate and extent of furrow progression is normal in control embryos injected with anti–GST antibody (Fig. 6 A). However, injections of the anti–Lva antibody cause severe furrowing defects (Fig. 6 A). At ∼50 min into cycle 14, furrow progression is clearly inhibited near the site of injection, where the antibody is most concentrated (Fig. 6 A). Further from the injection site, furrowing occurs at a reduced rate. Approximately 60 min into cellularization, the embryo's surface near the site of injection begins to dimple, becoming pronounced 10 min later (Fig. 6 A). This effect is presumably due to the pulling force exerted by the contractile apparatus on the surface of the embryo where furrowing has failed. Dimpling was never observed in control embryos. To directly examine the effects of the anti–GST and anti–Lva antibodies on Golgi, some injected embryos were fixed and analyzed by immunofluorescence using the anti–p120 cis-Golgi antibody. In control anti–GST antibody injected embryos, a normal distribution of p120 is observed (Fig. 6 B, left). However, in embryos injected with the concentrated anti–Lva antibody, the normal punctate distribution of p120 is not observed and instead p120 is found throughout the apical cytoplasm (Fig. 6 B, right). When the concentrated anti–Lva antibody is Cy5 tagged, the Lva protein can be seen to disperse within minutes of the injection, and the residual Lva protein left associated with some Golgi show them broken down and sessile (data not shown).

Bottom Line: Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated.Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190.We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, Sinsheimer Labs, University of California at Santa Cruz, Santa Cruz, California 95064, USA. sisson@darwin.ucsc.edu

ABSTRACT
Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

Show MeSH
Related in: MedlinePlus