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Investigating the role of an anti-mitotic soybean peptide (lunasin) in plant cell cycle

Investigating the role of an anti-mitotic soybean peptide (lunasin) in plant cell cycle. Mark Jesus M. Magbanua. UCD Plant Biology Graduate Group Student Seminar Series Winter 2003. Lunasin is a small subunit of a 2S albumin storage protein in soybeans. cDNA Gm2s-1 encodes :.

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Investigating the role of an anti-mitotic soybean peptide (lunasin) in plant cell cycle

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  1. Investigating the role of an anti-mitotic soybean peptide (lunasin) in plant cell cycle Mark Jesus M. Magbanua UCD Plant Biology Graduate Group Student Seminar Series Winter 2003

  2. Lunasin is a small subunit of a 2S albumin storage protein in soybeans cDNA Gm2s-1 encodes : signal peptide (21aa) small subunit (43 aa) linker (17 aa) large subunit (77aa) putative NLS lunasin RGDDDDDDDDD

  3. Lunasin disrupts bacterial cell division similar to an FtsZ (prokaryotic microtubule) mutation = septations Lunasin Lunasin-del (no poly A-tail) Induced expression of (A) lunasin and (B) lunasin-del in transformed DH5E. coli. (Galvez and de Lumen, Nature Biotech, 1999)

  4. Lunasin arrest cell division in mammalian cells Positive linear correlation between transfection efficiency and the proportion of lunasin-transfected murine hepatoma cells arrested at G2/M in 12 independent transfections. (Galvez and de Lumen, Nature Biotech, 1999)

  5. Lunasin induces apoptosis in mammalian cells Lunasin-del TUNEL positive cell DAPI Lunasin-GFP Transient expression of lunasin-del (1) and lunasin (2-4) in Human breast cancer cells (MCF-7). (Galvez and de Lumen, Nature Biotech, 1999)

  6. Lunasin-expressing cells produces elongated and misaligned spindle fibers Lunasin-del Indirect immunofluorescence (green) of microtubules in (A) lunasin-del and (B–D) lunasin-transfected murine hepatoma cells. (Galvez and de Lumen, Nature Biotech 1999)

  7. Lunasin preferentially binds to histones histones MT Associated Protein2 Solid-phase immunobinding assay for synthetic lunasin and lunasin-del peptides to (A) histones and (B) MAP2. (Galvez and de Lumen, Nature Biotech, 1999)

  8. GFP lunasin-frameshift in a human cell line GFP lunasin in a human cell line GFP lunasin in a mouse cell line Kinetochore formation is disrupted in mammalian cells expressing lunasin Double indirect immunofluorescence of kinetochore proteins and lunasin using CREST human antisera and lunasin polyclonal antisera, respectively (Galvez and de Lumen, Nature Biotech, 1999)

  9. 10 aa RGDDDDDDDDD Chromobinding motif Lunasin has a chromobinding domain found in other chromobinding proteins (Galvez et al., Cancer Research, 2001)

  10. Early seed development in angiosperms 1. Cell division (globular to torpedo stages) 2. Cell expansion a. massive synthesis of storage proteins b. increase in genomic DNA content = endoreduplication

  11. endoreduplication 24C 8C 4C 4C DNA content 2C 2C mitosis 2C 2C 2C time Endoreduplication in plants No endoreduplication in gametes, guard cells and the meristematic regions

  12. Lunasin is found in storage parenchyma cells and co-localizes with endoreduplicated DNA Expression pattern of Lunasin Gm2s-1 Endoreduplication Immunolocalization of Lunasin 40X 100X 40X Histochemical stain Lunasin immunostain Lunasin immunostain (arrows=endoreduplicated DNA) (arrows=lunasin co-localizing with endoreduplicated DNA) (Galvez et al. unpublished results)

  13. A B D E CCS52 Control Antimitotic gene, CCS52, induces cell enlargement and endoreduplication in fission yeast Overexpression of ccs52 in S.pombe results in endoreduplication and cell enlargement (Cebolla et al., EMBO, 1999)

  14. lunasin lunasin ? Plant cell cycle Seed growth and development Endoreduplication Mammalian cell cycle Eukaryotic Cell Cycle Is lunasin involved in the control of cell cycling during soybean seed development ? If so, is it involved in the induction of DNA endoreduplication and cell enlargement in the soybean cotyledon?

  15. When lunasin was cloned and expressed as a green fluorescent protein fusion in several mammalian cell lines, it inhibited mitosis by disrupting kinetochore function. Alfredo F. Galvez, Benito O. de Lumen. 1999.A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells. Nature Biotechnology 17, 495 – 500. Does lunasin have an effect on kinetochore function in other systems, e.g. yeast ?

  16. CFIII CFIII CFIII SUP11 HIS3 SUP11 HIS3 SUP11 HIS3 ade2-101 ade2-101 Visual Assay for Chromosome Stability YPH1015 YPH1161 YPH1015 -GFP CFIII CFIII SUP11 HIS3 SUP11 HIS3 ade2-101 ade2-101 ade2-101 skp1-4 LEU2 GFP-lunasin LEU2 Vector: pUD108 galactose-inducible promoter 10-5 10-2 ? Koshland et al., 1985 Connelly and Hieter, 1996

  17. YPH1015 YPH1161 Empty vector GFP-lunasin-fs GFP-lunasin-del GFP-lunasin YPH1015 YPH1161 Empty vector GFP-lunasin-fs GFP-lunasin-del GFP-lunasin raf raf/gal raf raf/gal raf raf/gal raf raf/gal raf raf/gal Test for Loss of Chromosome Selecting for both CFIII and vector Grow in Leu- His- minimal media + dextrose or raffinose to log phase Spin down and resuspend at 0.4x 107 cells/ml in Leu- minimal media + carbon source Allow loss of CFIII while selecting for vector and induce expression with gal Grow at 30°C until ~3.2 x 107 (3 doublings) Dilute and plate a known volume or known # of cells/ml on YPD Observe growth after 2 days and count red colonies Expected results for genes that disrupt chromosome segregation uninduced gene expression induced gene expression

  18. A B C B D E Visual assay for chromosome stability show that GFP lunasin does not cause increased rate of chromosome loss empty vector lunasin-del lunasin-fs lunasin positive control

  19. GFP lunasin has no effect on yeast kinetochore function a b c d YPH1015 a) empty vector pUD108 b) pUD108 GFP-lunasin-frameshift mutant c) pUD108 GFP-lunasin-deleted aspartic acid tail d) pUD108 GFP-lunasin - = uninduced + = induced Percent red colonies = number of red colonies/the number of colonies counted

  20. Viability is not reduced in yeast expressing GFP lunasin a b c d YPH1015 a) empty vector pUD108 b) pUD108 GFP-lunasin-frameshift mutant c) pUD108 GFP-lunasin-deleted aspartic acid tail d) pUD108 GFP-lunasin - = uninduced + = induced Percent viability = number of colonies counted/number of yeast cells plated

  21. Western blot analysis shows that lunasin is expressed as a GFP fusion protein in YPH1015 1 2 3 4 45 31 Lane 1 - empty vector pUD108 Lane 2 - pUD108 GFP-lunasin-frameshift mutant Lane 3 - pUD108 GFP-lunasin with deleted poly-aspartic acid tail Lane 4 - pUD108 GFP lunasin (33.5 kDa: GFP 29 kDa + lunasin 4.5 kDa). The blot is probed by a monoclonal antibody to lunasin at dilution 1:2000.

  22. Untagged lunasin does not cause increased rate of chromosome loss pUD108 pUD108 lunasin YPH1161 empty vector control positive control

  23. Subcellular localization of GFP lunasin in yeast GFP lunasin in a mammalian cell GFP-lunasin fs composite DAPI GFP-lunasin composite DAPI

  24. Particle bombardment of tobacco BY-2 cell suspension 5 to 10 µg DNA was used to transfect 3-day old tobacco BY-2 suspension cells 35S GFP lunasin

  25. Subcellular localization of GFP lunasin in tobacco BY-2 cells lunasin lunasin fs lunasin del

  26. GFP fusion protein expression 24 hr after particle bombardment lunasin lunasin fs

  27. GFP fusion protein expression 48 hr after particle bombardment lunasin lunasin fs

  28. Percentage of GFP expressing cells that underwent cell division

  29. Conclusions • Lunasin does not affect kinetochore function in budding yeast. • Lunasin GFP fusion protein is localized in both plant cytoplasm and the nucleus. • Transient expression of lunasin in plants does not conclusively show an effect on cell division.

  30. RB 35 S GFP lunasin BAR LB RB 35 S GFP lunasin-del BAR LB • Stable expression lunasin in plants • Constitutive expression in Arabidopsis • Arabidopsis expression in collaboration with the Britt Lab • 2. Inducible –dexamethasone inducible system • Tobacco BY-2 inducible expression in collaboration with the Liu lab

  31. lunasin ? lunasin lunasin ? Plant cell cycle Seed growth and development Endoreduplication Mammalian cell cycle Eukaryotic Cell Cycle

  32. Stals, H. and D. Inze. 2001. When plant cells decide to divide. TRENDS in Plant Science 6:8 359-364. Questions for future research:  What triggers cell division?  How do plant hormones communicate with the core cell-cycle machinery? What causes dividing cells to exit the cell cycle, to elongate and differentiate? Which signals convert a mitotic cell cycle into an endoreduplicating cell cycle?

  33. Endoreduplication levels in maize endosperm correlates with grain yield (Cavallini et al., 1995).

  34. The R. L. Rodriguez Laboratory Undergraduate students: Kevin Stoffel Sundeep Bhullar Visiting Scientist: Azadeh Kheirolomoom

  35. Collaborators: Alfredo Galvez, FilGen Biosciences Inc. Benito de Lumen, Nutrition-UC Berkeley The Kaplan Laboratory Thesis committee members: Anne Britt and Bo Liu Special thanks to: Jung-Youn Lee (Lucas Lab) Julie Lee (Liu Lab) Monica Brenni (Kaplan Lab) Joanna Friesner (Britt Lab) Eli Hefner (Britt Lab) DAVIS LADIES AUXILIARY

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