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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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sumo is required for normal syncytial nuclear cycles.A) A wild type syncytial blastoderm embryo in metaphase. B–E) Representative nuclear cycle defects in DAPI stained sumo04493 GLC embryos. DAPI staining revealed multiple cell cycle defects in sumo04493 GLC embryos. Panel B shows a sumo04493 mutant embryo, while B′ and B″ are magnified views of two regions of the embryo in B. The arrow in B′ points to an abnormally large cluster of chromosomes, indicating polyploidy, and the arrow in B″ points to a prominent chromosome bridge. C) Abnormal chromosomal organization. The arrow in D highlights a possible cohesion defect. The left arrow in E points to a cluster of hypercondensed chromosomes, and the right arrow points to chromosome fragments. F) Frequency of cell cycle defects in sumo and ubc9 mutant GLC embryos.
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pone-0005905-g004: sumo is required for normal syncytial nuclear cycles.A) A wild type syncytial blastoderm embryo in metaphase. B–E) Representative nuclear cycle defects in DAPI stained sumo04493 GLC embryos. DAPI staining revealed multiple cell cycle defects in sumo04493 GLC embryos. Panel B shows a sumo04493 mutant embryo, while B′ and B″ are magnified views of two regions of the embryo in B. The arrow in B′ points to an abnormally large cluster of chromosomes, indicating polyploidy, and the arrow in B″ points to a prominent chromosome bridge. C) Abnormal chromosomal organization. The arrow in D highlights a possible cohesion defect. The left arrow in E points to a cluster of hypercondensed chromosomes, and the right arrow points to chromosome fragments. F) Frequency of cell cycle defects in sumo and ubc9 mutant GLC embryos.

Mentions: The SUMO pathway has been shown to be required for cell cycle progression in other organisms [59]. Consistent with this, our proteomic analysis found proteins involved in cell cycle regulation to be significantly over-represented among SUMO conjugates in the early Drosophila embryo (Table 2, Table S6), and moreover our findings significantly expands the list of know sumoylated cell cycle regulators. To determine if the lethality caused by a reduced maternal supply of SUMO is due to cycling defects, 0- to 3-hour wild-type and sumo04493 GLC embryos were stained with DAPI to visualize DNA. During the initial stage of Drosophila embryogenesis, 13 nuclear cleavage cycles occur rapidly and synchronously in a syncytium (Figure 4A). We observed that over 50% of the sumo04493 GLC embryos exhibited a broad spectrum of nuclear cycle defects, including irregular size and distribution of nuclei, asynchronous nuclear division, abnormal interphase chromosome structure, overly condensed chromosomes, loss of sister chromatid cohesion during metaphase, polyploidy, chromosome clustering, fragmentation, and chromosome bridges (Figure 4B–E). Multiple nuclear division defects were often observed in a single embryo (Figure 4B). We also observed similar, although somewhat less penetrant, nuclear cleavage cycle defects in embryos resulting from GLC of a ubc9 (the SUMO conjugating enzyme) hypomorphic allele [12], semi118 (Figure 4F). The diverse cycling defects observed in the sumo and ubc9 GLC embryos indicate broad involvement of SUMO in multiple stages of the nuclear cycle, and are consistent with our proteomic analysis showing a significant enrichment in SUMO targets with cell cycle functions.


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)

sumo is required for normal syncytial nuclear cycles.A) A wild type syncytial blastoderm embryo in metaphase. B–E) Representative nuclear cycle defects in DAPI stained sumo04493 GLC embryos. DAPI staining revealed multiple cell cycle defects in sumo04493 GLC embryos. Panel B shows a sumo04493 mutant embryo, while B′ and B″ are magnified views of two regions of the embryo in B. The arrow in B′ points to an abnormally large cluster of chromosomes, indicating polyploidy, and the arrow in B″ points to a prominent chromosome bridge. C) Abnormal chromosomal organization. The arrow in D highlights a possible cohesion defect. The left arrow in E points to a cluster of hypercondensed chromosomes, and the right arrow points to chromosome fragments. F) Frequency of cell cycle defects in sumo and ubc9 mutant GLC embryos.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2692000&req=5

pone-0005905-g004: sumo is required for normal syncytial nuclear cycles.A) A wild type syncytial blastoderm embryo in metaphase. B–E) Representative nuclear cycle defects in DAPI stained sumo04493 GLC embryos. DAPI staining revealed multiple cell cycle defects in sumo04493 GLC embryos. Panel B shows a sumo04493 mutant embryo, while B′ and B″ are magnified views of two regions of the embryo in B. The arrow in B′ points to an abnormally large cluster of chromosomes, indicating polyploidy, and the arrow in B″ points to a prominent chromosome bridge. C) Abnormal chromosomal organization. The arrow in D highlights a possible cohesion defect. The left arrow in E points to a cluster of hypercondensed chromosomes, and the right arrow points to chromosome fragments. F) Frequency of cell cycle defects in sumo and ubc9 mutant GLC embryos.
Mentions: The SUMO pathway has been shown to be required for cell cycle progression in other organisms [59]. Consistent with this, our proteomic analysis found proteins involved in cell cycle regulation to be significantly over-represented among SUMO conjugates in the early Drosophila embryo (Table 2, Table S6), and moreover our findings significantly expands the list of know sumoylated cell cycle regulators. To determine if the lethality caused by a reduced maternal supply of SUMO is due to cycling defects, 0- to 3-hour wild-type and sumo04493 GLC embryos were stained with DAPI to visualize DNA. During the initial stage of Drosophila embryogenesis, 13 nuclear cleavage cycles occur rapidly and synchronously in a syncytium (Figure 4A). We observed that over 50% of the sumo04493 GLC embryos exhibited a broad spectrum of nuclear cycle defects, including irregular size and distribution of nuclei, asynchronous nuclear division, abnormal interphase chromosome structure, overly condensed chromosomes, loss of sister chromatid cohesion during metaphase, polyploidy, chromosome clustering, fragmentation, and chromosome bridges (Figure 4B–E). Multiple nuclear division defects were often observed in a single embryo (Figure 4B). We also observed similar, although somewhat less penetrant, nuclear cleavage cycle defects in embryos resulting from GLC of a ubc9 (the SUMO conjugating enzyme) hypomorphic allele [12], semi118 (Figure 4F). The diverse cycling defects observed in the sumo and ubc9 GLC embryos indicate broad involvement of SUMO in multiple stages of the nuclear cycle, and are consistent with our proteomic analysis showing a significant enrichment in SUMO targets with cell cycle functions.

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