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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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The purification of Drosophila MMAPs. (A) This Coomassie-stained SDS-polyacrylamide gradient gel shows the proteins within different fractions from the purification: high-speed pellet (P100) and supernatant (S100, column load), F-actin–binding proteins (ABPs), and F-actin column flow through (F.T.). Lanes 1–4 were each loaded with 25 μg of total protein. ABPs do not sediment without MTs (lanes 5 and 6), but a small subset of ABPs do sediment with MTs (spin 1). The lane 8 pellet was rinsed, resuspended, and the MTs were resedimented (spin 2). Numbered arrows indicate the proteins that resediment with the MTs. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded. Molecular weight standards are indicated to the left. *Presumed protein degradation. (B) Immunoblots for MHC, 95F, and actin. Lanes are labeled and loaded as in A, lanes 5 and 6 correspond to spin 1 in A. (C) Immunoblots for the indicated MT-binding proteins, and tubulin. Each lane is labeled and loaded as in A. (D, left) Equivalent protein blots probed with antibodies to the indicated proteins. Each lane is labeled and loaded as in A. Coomassie-stained band numbers and molecular weights are indicated to the right. (Right) S100 (25 μg) protein blot probed with affinity-purified rabbit anti–Lva antibody. Molecular weight standards are indicated.
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Figure 1: The purification of Drosophila MMAPs. (A) This Coomassie-stained SDS-polyacrylamide gradient gel shows the proteins within different fractions from the purification: high-speed pellet (P100) and supernatant (S100, column load), F-actin–binding proteins (ABPs), and F-actin column flow through (F.T.). Lanes 1–4 were each loaded with 25 μg of total protein. ABPs do not sediment without MTs (lanes 5 and 6), but a small subset of ABPs do sediment with MTs (spin 1). The lane 8 pellet was rinsed, resuspended, and the MTs were resedimented (spin 2). Numbered arrows indicate the proteins that resediment with the MTs. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded. Molecular weight standards are indicated to the left. *Presumed protein degradation. (B) Immunoblots for MHC, 95F, and actin. Lanes are labeled and loaded as in A, lanes 5 and 6 correspond to spin 1 in A. (C) Immunoblots for the indicated MT-binding proteins, and tubulin. Each lane is labeled and loaded as in A. (D, left) Equivalent protein blots probed with antibodies to the indicated proteins. Each lane is labeled and loaded as in A. Coomassie-stained band numbers and molecular weights are indicated to the right. (Right) S100 (25 μg) protein blot probed with affinity-purified rabbit anti–Lva antibody. Molecular weight standards are indicated.

Mentions: The initial step of the protein purification was to isolate actin-binding proteins by F-actin affinity chromatography. A high-speed supernatant of a Drosophila embryo extract was passed over an F-actin column, and after extensive washing, the column was eluted with buffer containing 1 mM ATP and 0.5 M KCl. After SDS-PAGE, Coomassie staining revealed ∼50 polypeptide bands in the ABP fraction (Fig. 1 A, lane 3). Together, the ABPs comprise 0.5–1.0% of the total soluble protein passed over the F-actin column. Immunoblots demonstrate that two known ABPs, MHC and 95F, an unconventional Myosin VI, are highly enriched on the F-actin column, consistent with previous results (Fig. 1 B, lanes 2 and 3) (Miller et al. 1989). Conversely, the MT-binding proteins kinesin heavy chain, centrosomal protein (CP) 190, CP60, p150Glued, and dynein, as well as tubulin, do not bind significantly to the F-actin column, indicating selective binding (Fig. 1 C, lanes 2 and 3; dynein 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)

The purification of Drosophila MMAPs. (A) This Coomassie-stained SDS-polyacrylamide gradient gel shows the proteins within different fractions from the purification: high-speed pellet (P100) and supernatant (S100, column load), F-actin–binding proteins (ABPs), and F-actin column flow through (F.T.). Lanes 1–4 were each loaded with 25 μg of total protein. ABPs do not sediment without MTs (lanes 5 and 6), but a small subset of ABPs do sediment with MTs (spin 1). The lane 8 pellet was rinsed, resuspended, and the MTs were resedimented (spin 2). Numbered arrows indicate the proteins that resediment with the MTs. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded. Molecular weight standards are indicated to the left. *Presumed protein degradation. (B) Immunoblots for MHC, 95F, and actin. Lanes are labeled and loaded as in A, lanes 5 and 6 correspond to spin 1 in A. (C) Immunoblots for the indicated MT-binding proteins, and tubulin. Each lane is labeled and loaded as in A. (D, left) Equivalent protein blots probed with antibodies to the indicated proteins. Each lane is labeled and loaded as in A. Coomassie-stained band numbers and molecular weights are indicated to the right. (Right) S100 (25 μg) protein blot probed with affinity-purified rabbit anti–Lva antibody. Molecular weight standards are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169433&req=5

Figure 1: The purification of Drosophila MMAPs. (A) This Coomassie-stained SDS-polyacrylamide gradient gel shows the proteins within different fractions from the purification: high-speed pellet (P100) and supernatant (S100, column load), F-actin–binding proteins (ABPs), and F-actin column flow through (F.T.). Lanes 1–4 were each loaded with 25 μg of total protein. ABPs do not sediment without MTs (lanes 5 and 6), but a small subset of ABPs do sediment with MTs (spin 1). The lane 8 pellet was rinsed, resuspended, and the MTs were resedimented (spin 2). Numbered arrows indicate the proteins that resediment with the MTs. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded. Molecular weight standards are indicated to the left. *Presumed protein degradation. (B) Immunoblots for MHC, 95F, and actin. Lanes are labeled and loaded as in A, lanes 5 and 6 correspond to spin 1 in A. (C) Immunoblots for the indicated MT-binding proteins, and tubulin. Each lane is labeled and loaded as in A. (D, left) Equivalent protein blots probed with antibodies to the indicated proteins. Each lane is labeled and loaded as in A. Coomassie-stained band numbers and molecular weights are indicated to the right. (Right) S100 (25 μg) protein blot probed with affinity-purified rabbit anti–Lva antibody. Molecular weight standards are indicated.
Mentions: The initial step of the protein purification was to isolate actin-binding proteins by F-actin affinity chromatography. A high-speed supernatant of a Drosophila embryo extract was passed over an F-actin column, and after extensive washing, the column was eluted with buffer containing 1 mM ATP and 0.5 M KCl. After SDS-PAGE, Coomassie staining revealed ∼50 polypeptide bands in the ABP fraction (Fig. 1 A, lane 3). Together, the ABPs comprise 0.5–1.0% of the total soluble protein passed over the F-actin column. Immunoblots demonstrate that two known ABPs, MHC and 95F, an unconventional Myosin VI, are highly enriched on the F-actin column, consistent with previous results (Fig. 1 B, lanes 2 and 3) (Miller et al. 1989). Conversely, the MT-binding proteins kinesin heavy chain, centrosomal protein (CP) 190, CP60, p150Glued, and dynein, as well as tubulin, do not bind significantly to the F-actin column, indicating selective binding (Fig. 1 C, lanes 2 and 3; dynein 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