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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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Lva associates with Spectrins and CLIP190. Immunoblots show (A) gel filtration elution profiles for the indicated proteins in the S100 and MMAP fractions. Fraction numbers and the elution peaks for native molecular weight standards are shown at the top. (B) Elution profiles for the indicated proteins that rebind an F-actin column. Lane headings indicate F-actin column load (ABPs), F-actin column flow through (F.T.), and elution fraction numbers. Identical results for each protein were also obtained with the MMAP fraction (data not shown). (C) A mock IP performed with BSA, and IPs performed with anti–Lva, anti–α-Spectrin, and anti–CLIP190 antibodies on the MMAP fraction. Proteins detected by immunoblot are indicated to the left. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded.
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Figure 3: Lva associates with Spectrins and CLIP190. Immunoblots show (A) gel filtration elution profiles for the indicated proteins in the S100 and MMAP fractions. Fraction numbers and the elution peaks for native molecular weight standards are shown at the top. (B) Elution profiles for the indicated proteins that rebind an F-actin column. Lane headings indicate F-actin column load (ABPs), F-actin column flow through (F.T.), and elution fraction numbers. Identical results for each protein were also obtained with the MMAP fraction (data not shown). (C) A mock IP performed with BSA, and IPs performed with anti–Lva, anti–α-Spectrin, and anti–CLIP190 antibodies on the MMAP fraction. Proteins detected by immunoblot are indicated to the left. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded.

Mentions: To assess whether Lva interacts with other proteins, we first determined the native size of Lva compared with other MMAPs. The S100 and the final protein (MMAP) fraction were passed separately over a gel filtration column, and fractions were assayed by immunoblot. Because α-Spectrin has been previously shown to copurify with β- and βH-Spectrin in two stable heterotetrameric complexes (α2β2 and α2βH2, respectively) (Dubreuil et al. 1990) and coimmunoprecipitates with β- and βH-Spectrin (Fig. 3 C), we will only present data for α-Spectrin for simplicity. Immunoblots show that in both the S100 and the MMAP fraction, Lva, CLIP190, αβ-, and αβH-Spectrin coelute from the column with native molecular weights larger then their predicted molecular weights, indicating that each protein exists in large, stable complexes (Fig. 3 A). By comparison, Anillin and KLP3A are initially present in large complexes that dissociate over the course of the purification (Fig. 3 A). The native molecular weight of Anillin in the final fraction is comparable with that observed for baculovirus-expressed, full-length frog Anillin (A. Straight, personal communication). Unlike the other proteins, most of 95F does not exist in a large complex in the S100 (Fig. 3 A).


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)

Lva associates with Spectrins and CLIP190. Immunoblots show (A) gel filtration elution profiles for the indicated proteins in the S100 and MMAP fractions. Fraction numbers and the elution peaks for native molecular weight standards are shown at the top. (B) Elution profiles for the indicated proteins that rebind an F-actin column. Lane headings indicate F-actin column load (ABPs), F-actin column flow through (F.T.), and elution fraction numbers. Identical results for each protein were also obtained with the MMAP fraction (data not shown). (C) A mock IP performed with BSA, and IPs performed with anti–Lva, anti–α-Spectrin, and anti–CLIP190 antibodies on the MMAP fraction. Proteins detected by immunoblot are indicated to the left. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded.
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Figure 3: Lva associates with Spectrins and CLIP190. Immunoblots show (A) gel filtration elution profiles for the indicated proteins in the S100 and MMAP fractions. Fraction numbers and the elution peaks for native molecular weight standards are shown at the top. (B) Elution profiles for the indicated proteins that rebind an F-actin column. Lane headings indicate F-actin column load (ABPs), F-actin column flow through (F.T.), and elution fraction numbers. Identical results for each protein were also obtained with the MMAP fraction (data not shown). (C) A mock IP performed with BSA, and IPs performed with anti–Lva, anti–α-Spectrin, and anti–CLIP190 antibodies on the MMAP fraction. Proteins detected by immunoblot are indicated to the left. Comparable amounts of each matched supernatant (S) and pellet (P) were loaded.
Mentions: To assess whether Lva interacts with other proteins, we first determined the native size of Lva compared with other MMAPs. The S100 and the final protein (MMAP) fraction were passed separately over a gel filtration column, and fractions were assayed by immunoblot. Because α-Spectrin has been previously shown to copurify with β- and βH-Spectrin in two stable heterotetrameric complexes (α2β2 and α2βH2, respectively) (Dubreuil et al. 1990) and coimmunoprecipitates with β- and βH-Spectrin (Fig. 3 C), we will only present data for α-Spectrin for simplicity. Immunoblots show that in both the S100 and the MMAP fraction, Lva, CLIP190, αβ-, and αβH-Spectrin coelute from the column with native molecular weights larger then their predicted molecular weights, indicating that each protein exists in large, stable complexes (Fig. 3 A). By comparison, Anillin and KLP3A are initially present in large complexes that dissociate over the course of the purification (Fig. 3 A). The native molecular weight of Anillin in the final fraction is comparable with that observed for baculovirus-expressed, full-length frog Anillin (A. Straight, personal communication). Unlike the other proteins, most of 95F does not exist in a large complex in the S100 (Fig. 3 A).

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