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Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

Nie M, Xie Y, Loo JA, Courey AJ - PLoS ONE (2009)

Bottom Line: For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation.We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody.Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

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Roles for SUMO in embryonic and eggshell patterning.A) Follicle cell clones in the sumo04493 GLC egg chambers. Wild-type (Ore-R; top) and sumo04493 GLC egg chambers (bottom) were stained with SUMO antibodies (green) and DAPI (blue). The field in the lower panels contains a sumo04493/04493 clone resulting from FLP/FRT recombination (yellow arrows), heterozygous sumo04493/+ cells with a reduced level of SUMO (white arrows), and a sumo+/+ twin spot containing a level of SUMO comparable to that observed in Ore-R follicle cells (red arrows). B) A variety of anteroposterior (3 panels on the left) and dorsoventral (panel on the right) patterning defects were observed in sumo04493 mutant GLC embryos. Among the embryos that formed cuticles, 59% (n = 171) exhibited abnormal cuticular morphology. Top panel shows a wild type cuticle. C) Examples of ventralized eggshells of sumo04493 GLC embryos are shown in the three panels on the right. A wild type Ore-R embryo is shown in the leftmost panel.
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pone-0005905-g002: Roles for SUMO in embryonic and eggshell patterning.A) Follicle cell clones in the sumo04493 GLC egg chambers. Wild-type (Ore-R; top) and sumo04493 GLC egg chambers (bottom) were stained with SUMO antibodies (green) and DAPI (blue). The field in the lower panels contains a sumo04493/04493 clone resulting from FLP/FRT recombination (yellow arrows), heterozygous sumo04493/+ cells with a reduced level of SUMO (white arrows), and a sumo+/+ twin spot containing a level of SUMO comparable to that observed in Ore-R follicle cells (red arrows). B) A variety of anteroposterior (3 panels on the left) and dorsoventral (panel on the right) patterning defects were observed in sumo04493 mutant GLC embryos. Among the embryos that formed cuticles, 59% (n = 171) exhibited abnormal cuticular morphology. Top panel shows a wild type cuticle. C) Examples of ventralized eggshells of sumo04493 GLC embryos are shown in the three panels on the right. A wild type Ore-R embryo is shown in the leftmost panel.

Mentions: Two independent P-insertions sumo alleles, termed sumo04493 and sumok01211, were subjected to phenotypic analysis. The P-insertion in both alleles is 20 bp upstream of the transcription start site, creating recessive lethal mutations with a lethal period before or during the early second larval instar. Evaluation of mRNA levels by RT-PCR in homozygous mutant larvae shows that the sumo04493 mutation leads to an approximately 5-fold decrease in the level of sumo transcripts (Figure S3). Antibody staining of mutant follicle cell clones (see below) also demonstrates a significant reduction in SUMO levels in mutant tissue (Figure 2A). sumo04493, sumok01211, and a sumo EMS allele (generated by Shanti Chandrashekaran, New Delhi, and obtained through Dr. Lawrence Marsh), which contains a serine-glycine sequence in place of the normal C-terminal di-glycine motif required for efficient conjugation of SUMO to its targets [6], [23] fail to complement one another.


Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

Nie M, Xie Y, Loo JA, Courey AJ - PLoS ONE (2009)

Roles for SUMO in embryonic and eggshell patterning.A) Follicle cell clones in the sumo04493 GLC egg chambers. Wild-type (Ore-R; top) and sumo04493 GLC egg chambers (bottom) were stained with SUMO antibodies (green) and DAPI (blue). The field in the lower panels contains a sumo04493/04493 clone resulting from FLP/FRT recombination (yellow arrows), heterozygous sumo04493/+ cells with a reduced level of SUMO (white arrows), and a sumo+/+ twin spot containing a level of SUMO comparable to that observed in Ore-R follicle cells (red arrows). B) A variety of anteroposterior (3 panels on the left) and dorsoventral (panel on the right) patterning defects were observed in sumo04493 mutant GLC embryos. Among the embryos that formed cuticles, 59% (n = 171) exhibited abnormal cuticular morphology. Top panel shows a wild type cuticle. C) Examples of ventralized eggshells of sumo04493 GLC embryos are shown in the three panels on the right. A wild type Ore-R embryo is shown in the leftmost panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2692000&req=5

pone-0005905-g002: Roles for SUMO in embryonic and eggshell patterning.A) Follicle cell clones in the sumo04493 GLC egg chambers. Wild-type (Ore-R; top) and sumo04493 GLC egg chambers (bottom) were stained with SUMO antibodies (green) and DAPI (blue). The field in the lower panels contains a sumo04493/04493 clone resulting from FLP/FRT recombination (yellow arrows), heterozygous sumo04493/+ cells with a reduced level of SUMO (white arrows), and a sumo+/+ twin spot containing a level of SUMO comparable to that observed in Ore-R follicle cells (red arrows). B) A variety of anteroposterior (3 panels on the left) and dorsoventral (panel on the right) patterning defects were observed in sumo04493 mutant GLC embryos. Among the embryos that formed cuticles, 59% (n = 171) exhibited abnormal cuticular morphology. Top panel shows a wild type cuticle. C) Examples of ventralized eggshells of sumo04493 GLC embryos are shown in the three panels on the right. A wild type Ore-R embryo is shown in the leftmost panel.
Mentions: Two independent P-insertions sumo alleles, termed sumo04493 and sumok01211, were subjected to phenotypic analysis. The P-insertion in both alleles is 20 bp upstream of the transcription start site, creating recessive lethal mutations with a lethal period before or during the early second larval instar. Evaluation of mRNA levels by RT-PCR in homozygous mutant larvae shows that the sumo04493 mutation leads to an approximately 5-fold decrease in the level of sumo transcripts (Figure S3). Antibody staining of mutant follicle cell clones (see below) also demonstrates a significant reduction in SUMO levels in mutant tissue (Figure 2A). sumo04493, sumok01211, and a sumo EMS allele (generated by Shanti Chandrashekaran, New Delhi, and obtained through Dr. Lawrence Marsh), which contains a serine-glycine sequence in place of the normal C-terminal di-glycine motif required for efficient conjugation of SUMO to its targets [6], [23] fail to complement one another.

Bottom Line: For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation.We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody.Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

Show MeSH
Related in: MedlinePlus