Progress 09/01/02 to 08/31/08
Outputs
OUTPUTS: The growth, differentiation and development of all organisms are defined by precise molecular events governing cell division. A comprehensive understanding of these events is provided by studies on the chromosome and its protein, DNA and RNA components. Thus, in a collaborative approach, Professors Jack Girton, Kristen Johansen and Jorgen Johansen have continued to provide new information on the proteins that are critical components of the mitotic spindle. The Ambrosio lab is using the extensive genetics knowledge of Drosophila melanogaster to understand the role of D-Raf kinase in cell division. The laboratory of Professor Don Sakaguchi has maintained focus on understanding the mechanisms for controlling stem cell differentiation that would ultimately benefit the development of therapeutic strategies for tissue regeneration in the eye as well as other organs. Studies conducted in the Phil Becraft laboratory have continued to provide important insights into the mechanism of formation of the maize endosperm and Professor Don Beitz and collaborators have identified novel genes involved in the regulation of Mycobacterium mediated diseases in cows. The Marit Nilsen-Hamilton laboratory is developing novel tools to understand, in a quantitative sense, gene expression in vivo. The experiments described in this report included participation from graduate students and/or post-doctoral fellows under the guidance of the principal investigators (note that Dr. Jan Buss is no longer in the department of BBMB). The results from the scientific investigations described above were shared with the community through a variety of forums that included oral presentations, poster presentations and publications in peer-reviewed journals. These forums facilitate the widest possible dissemination of research knowledge with broad impact. PARTICIPANTS: Dr's. Girton, Nilsen-Hamilton, Johansen K, Johansen J, Beitz and Ambrosia are faculty in the Department of Biochemistry, Biophysics, and Molecular Biology (BBMB). Dr. Becraft and Dr. Sakaguchi are faculty members in the Department of Genetics, Development and Cell Biology (GDCB). Other participants include graduate students, post-doctoral fellows and P&S staff. TARGET AUDIENCES: Target audiences include the international scientific community, especially individuals working on questions involving the molecular genetic control of chromosome structure and /or cell division. Research findings of the last few years have been shared with peers in the field at various research meetings as outlined in previous reports and indicated below for this past year. Don Sakaguchi: Annual Meeting-The Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida (2008); Annual Meeting, Society for Bioelectric Signaling, San Diego, CA (2008); XVIII International Congress of Eye Research (ICER) Beijing, China (2008); Korean Society of Stem Cell Research. Abstract P-63 (2008). Kristen Johansen: Cold Spring Harbor Laboratory 6th Meeting on Dynamic Organization of Nuclear Function, Cold Spring Harbor, N.Y (2008); Iowa State University, Department of Genetics, Development & Cell Biology, Ames, IA (2008); American Society for Cell Biology Symposium, "The Spindle Matrix", San Francisco, CA (2008). Marit Nilsen-Hamilton: Marrakesh International Conference and Workshop on Mathematical Biology, Morocco - Plenary speaker; 2 seminars and panel discussion leader (2008); Washington D.C. Plenary speaker at DOE workshop (2008). Phil Becraft: The beginning and the end of aleurone development (2008, GDCB Departmental seminar); Endosperm development (2008, GDCB Departmental seminar); Genetic Analysis of maize kernel development (2008, Interdepartmental Genetics Seminar). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Investigations in the Sakaguchi group and their collaborators have produced new knowledge towards the understanding of the development of the retina, an important part of the eye for visual perception. In one project they have studied stem cells specifically associated with the development of the retina. They have examined the differences in the proteins expressed by cells destined to become the retina (retinal progenitor cells) and cells destined to become brain tissue (brain-progenitor cells) and have identified proteins that are unique to the retinal cell state. Such results are anticipated to provide fundamental knowledge to manipulate the fate of other types of cells to also become retinal cells and thus facilitate the development of therapy to replace retinal neurons lost to disease. In a second project, they have studied the cellular basis of the formation of the retina in a marsupial (opossum) as a system to explore the plasticity of neural stem cells for transplantations. In yet another project Sakaguchi and collaborators have examined the role of proteins that could potentially protect the retina from damage resulting from a decrease in the supply of oxygen. 2) The Johansen and Girton group have produced new knowledge on the role of some recently discovered molecules involved in chromosome organization and cell division. Thus, they have looked more closely at the JIL-1 histone H3S10 kinase in Drosophila. This is the protein whose key function is to regulate the modification (phosphorylations) if histone H3 in interphase chromosomes. They have determined that a specific region of this protein, towards the carboxy-terminal end, is important for regulating chromatin structure. In parallel, they have demonstrated that transcriptional defects in the absence of histone phosphorylations are a consequence of structural changes in chromatin. 3) Investigations in the Becraft laboratory have resulted in significant advances in our understanding of the development of the endosperm. Using both genetic, biochemical and cell biology based methods they have identified proteins that are involved in signaling pathways for differentiation of endosperm cells. These results have profound implications in developing plant genetic engineering methodologies to positively affect seed development for food and fuel.
Publications
- T. Dunn-Thomas, D. L. Dobbs, D.S. Sakaguchi, M.J. Young, V.G. Honavar, M.H. West Greenlee. (2008) Proteomic differentiation between murine retinal and brain-derived progenitor cells. Stem Cells and Development. 17(1) 119-132.
- Sakaguchi, D.S., Van Hoffelen, S.J., Greenlee, M.H.W., Harper, M.H., and Au, D. (2008) Cell birth and death in the retina of the Brazilian opossum, Monodelphis domestica. Brain Research. 1195C: 28-42.
- Ostojic, J., Syed, N.A., Hargrove, M.S., Trent, J.T. III, Kuehn, M.H., Kardon, R.H., Kwon, Y.H., Grozdanic, S.D., and Sakaguchi, D.S. (2008) Neuroglobin and Cytoglobin Distribution in the Anterior Segment: A Comparative Study of the Human and Dog Eye. Journal of Histology and Immunohistochemistry. 56: 863-872.
- Ostojic, J., Grozdanic, S.D., Syed, N.A., Hargrove, M.S., Trent, J.T. III, Kuehn, M.H., Kwon, Y.H., Kardon, R.H., and Sakaguchi, D.S. (2008) Patterns of Distribution of Oxygen Binding Globins, Neuroglobin and Cytoglobin in Human Retina (2008) Archives of Ophthalmology. 126(11): 1530-1536.
- Bao, X., Cai, W., Deng, H., Zhan, W., Krencik, R., Girton, J., Johansen, J., Johansen, K.M. (2008) The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting of chromatin. J. Biol.Chem. 283: 32741-50.
- Cai, W., Bao, X., Deng, H., Jin, Y., Girton, J., Johansen, J., Johansen, K.M. (2008) RNA polymerase-II mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila. Development 135: 2917-25.
- Girton, J.R., Johansen, K.M. (2008). Chromatin structure and the regulation of gene expression: the lessons of PEV in Drosophila. Adv. Genet. 61: 1-43.
- Karcher, E.L., Bayles, D.O., Bannatine, J.P., Beitz, D.C. and Stabel, J.R. (2008) Osteopentin: a novel cytokine involved in the regulation of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle. J. Dairy Sci. 91: 3079-91
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In collaboration with K. Johansen and J Johansen, Girton has conducted a series of biochemical and molecular genetic experiments of a series of genes and gene products in Drosophila melanogaster. These genes and products are essential for normal cell division and cell growth. The experiments were designed to discover basic information about the function of these genes and their role in controlling two key processes: formation and function of the mitotic spindle during cell division and organization of DNA and chromatin proteins in interphase chromosomes. The Nilsen-Hamilton lab has been studying an acute phase protein that is produced in large amounts by reproductive tissues during involution. The protein plays a role as a bacteriostatic agent and also appears to be anti-inflammatory. The goal of the group is to understand the physiological role of this protein. The Ambrosio lab has conducted a series of molecular genetic experiments to define the regulatory role of protein
sequences of D-Raf in Drosophila melanogaster. The D-Raf gene is essential for the transfer of developmental information important for normal cell division, growth and differentiation. The experiments were designed to discover basic information about the function of N-terminal sequences, whether they participate in the positive and/or negative regulation of the D-raf protein. They have used deletion and over-expression analysis to accomplish this goal. The Becraft lab has conducted a series of molecular genetic experiments on genes in Zea mays. These genes and products are essential for normal grain development. The experiments were designed to discover basic information about the function of these genes and their role in controlling the processes involved in grain development. To disseminate the information, the described work has been published or is in preparation for publication in international journals.
PARTICIPANTS: Dr. Jack R. Girton (coPI) and collaborators Dr. Kristen Johansen and Dr. Jorgen Johansen, Department of Biochemistry, Biophysics, and Molecular Biology. Dr. Marit Nilsen-Hamilton (PI) Department of Biochemistry, Biophysics, and Molecular Biology. Wei Zhao, PhD student, Molecular Cellular and Developmental Biology Program Dr. Linda Ambrosio (PI) Department of Biochemistry, Biophysics, and Molecular Biology. Dr. Philip W. Becraft (PI) Genetics, Development and Cell Biology Department, Antony Chettoor, PhD student, Interdepartmental Plant Physiology Major Lixun Su, PhD student, Interdepartmental Plant Physiology Major Joonbae Seo, MS student, Interdepartmental Plant Physiology Major
TARGET AUDIENCES: Target audiences include the international scientific community, especially individuals working on questions involving the molecular genetic control of chromosome structure, signal transduction, plant development, gene expression, cell differentiation, cell division and the inflammatory response.
Impacts
The Girton group produced a body of new knowledge about the molecular mechanisms of cell division and chromosome organization in cells. They also produced a collection of unique genetic mutant strains. They discovered that the JIL-1 gene product, a tandem kinase, has a key function in regulating the modification (phosphorylation) of histone H3 in interphase chromosomes. They found that a collection of genes named Skeletor, Chromator, Megator, and East, which participate in the formation of a spindle matrix, physically interact with each other during spindle matrix function and with spindle microtubules in a way that is essential for normal chromosome segregation and successful cell division. Another key outcome is the generation of a large collection of over 100 unique genetic strains containing new mutations they have generated as well as new combinations of genes or cloned gene constructs. The Nilsen-Hamilton lab produced new information about gene expression during
inflammation and demonstrated that the Lcn2 gene is expressed in large amounts in the lung and the gastrointestinal tract during the inflammatory response and that Lcn2 expression in macrophages is increased in response to LPS. The Ambrosio group concluded that within the N-terminal region of D-Raf a 60 amino acid sequence has been conserved in other species of Drosophila, as well as higher animals, including those of agricultural importance and humans. They termed this region CRN for Conserved Region N-terminal and found that N-terminal sequences of D-Raf including CRN are necessary to achieve its full activation and function. The Becraft group produced new knowledge about the molecular genetic mechanisms of cell fate specification in the maize endosperm. They identified a new mutant with extra aleurone layers, which is epistatic to other mutants that lack aleurone. This significantly impacts existing models for how aleurone is specified. They also cloned a gene required for the
differentiation of basal transfer cells, required to transport solutes from maternal tissues to the developing seeds. A second project involves studying the transcriptional regulation of the viviparous1 gene, which controls the developmental decision between seed maturation or seed germination. They identified two proteins that bind different elements of the vp1 promoter and are studying the function of these proteins. In a third project, they developed a high throughput gene cloning method that they hope to apply to the analysis of essential genes for maize grain development by cloning seed lethal mutants.
Publications
- Ding, Y., Rath U., Qi H., Zhu L., Girton J., Johansen J. and Johansen K. M. (2007) The spindle matrix complex protein, Chromator, is required for proper microtubule spindle formation and mitosis in Drosophila neuroblasts. Mol. Biol. Cell 18.
- Deng, H., X. Bao, W. Cai, M. J. Blacketer, A. S. Belmont, J. Girton, J. Johansen and K. M. Johansen (2007) Remodeling of interphase chromatin structure caused by ectopic histone H3S10 phosphorylation. Mol. Biol. Cell 18.
- Bao, X., Deng H., Cai W., Girton J., Johansen J. and Johansen K. M. (2007) Structure/function Analysis of the JIL-1 histone H3S10 tandem kinase. Mol. Biol. Cell 18.
- Bao X., Girton J., Johansen J., and Johansen K. M. 2007 The lamin DmoAri3 allele acts as an enhancer of position effect variegation at the wm4 locus in Drosophila. Genetica 129, 339-342
- Bao X., Deng H., Johansen J., Girton J., and Johansen K. M. 2007. Loss-of-function alleles of the JIL-1 histone H3S10 kinase enhance position-effect-variegation at pericentric sites in Drosophila heterochromatin. Genetics 176, 1355-8
- Deng H., Bao X., Zhang W., Girton J., Johansen J. and Johansen K. M. 2007 Reduced levels of Su(var)3-9 but not Su(var)2-5(HP1) counteract the effects on chromatin structure and viability of loss of JIL-1 histone H3S10 kinase activity. Genetics 177, 1-9
- Becraft, P. W. (2007). Aleurone cell development. In "Endosperm - Development and Molecular biology" (O.-A. Olsen, Ed.), pp. 45-56. Springer.
- Sunil V. R., Patel K. J., Nilsen-Hamilton M., Heck D. E., Laskin J. D., Laskin D. L.(2007) Acute endotoxemia is associated with upregulation of lipocalin 24p3/Lcn2 in lung and liver. Exp Mol Pathol. 83:177-187
- Soares M. J., Khorshed Alam S. M., Duckworth M. L., Horseman N. D., Konno T., Linzer D. H., Maltais L. J., Nilsen-Hamilton M., Shiota K., Smith J. R., Wallis M. (2007) A standardized nomenclature for the mouse and rat prolactin superfamilies. Mammalian Genome 18, 154-6.
- Cao, X., Costa, L. M., Biderre-Petit, C., Kbhaya, B., Dey, N., Perez, P., McCarty, D. R., Gutierrez-Marcos, J. F., and Becraft, P. W. (2007). Abscisic acid and stress signals induce Viviparous1 expression in seed and vegetative tissues of maize. Plant Physiol. 143, 720-731.
- Settles, A. M., Holding, D., Tan, B., Latshaw, S., Liu, J., Suzuki, M., Li, L., O'Brien, B., Fajardo, D., Wroclawska, E., Tseung, C.-W., Lai, J., Hunter, C., Avigne, W., Baier, J., Messing, J., Hannah, L. C., Koch, K., Becraft, P., Larkins, B., and McCarty, D. (2007). Sequence-indexed mutations in maize using the UniformMu transposon-tagging population. BMC Genomics 8, 116.
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Progress 01/01/05 to 12/31/05
Outputs
Area 1) Progress has been made towards understanding the mechanisms of signal transduction in plants from the CRINKLY4 serine/threonine receptor-like kinase. This protein kinase controls an array of developmental processes in the plant and endosperm. In the current project period the CRINKLY4 homologs of Arabidopsis were analyzed by reverse genetics using T-DNA insertion mutants. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of maize CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The five Arabidopsis genes are differentially expressed in various tissues. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function. These studies have shed new light on the CRINKLY4 signaling
system, revealing that multiple pathways may be involved in generating the same signals. Area 2) Significant progress has been made in the development of novel biomolecules that can be used to indicate the presence of specific small molecules present in cells in vivo. The work has used aptamers as such biosensors. Aptamers are unique nucleic acids with the ability to participate in at least two different types of three-dimensional structure. The project made use of this structural flexibility to develop a biosensor that controls the ability of the aptamer to bind to its target molecule. The results have shown that aptamers that can be regulated by a specific nucleic sequence such as in an mRNA have potential for many in vivo applications including regulating a particular enzyme or signal transduction pathway or imaging gene expression in vivo. Area 3) We have continued to make progress regarding how chromatin function is controlled by the JIL-1 tandem kinase. JIL-1 is an essential
kinase and mutational analysis has shown that it is the predominant kinase regulating histone H3 Ser10 phosphorylation in the interphase nucleus. Recent progress in the project demonstrated that the Drosophila nuclear lamin Dm0 directly interacts with the COOH-terminal domain of JIL-1. The results suggest that JIL-1 kinase activity is required to maintain nuclear morphology and integrity of nurse cells during oogenesis and that this function may be linked to molecular interactions with lamin Dm0. A broad conclusion from the recent progress in the project is that JIL-1 is a coordinating factor that affects both transcriptional control and nuclear architecture.
Impacts
One of most important cellular actions is cell division. This is controlled by a complex of internal and external signals. These studies are expected to improve our understanding of how cell division is regulated, and other studies hold tremendous potential for the treatment of a variety of degenerative disorders of the nervous system. They are expected to open new uses of stem cells beyond the still-unattained goal of regeneration of specific tissue types, into a broader role as cells to support repair in many degenerative diseases. Our studies of uterocalin/FREB are expected to lead to a better understanding of the molecular basis of tissue damage in inflammation as occurs in mastitis and Johne's disease.
Publications
- Bao X, Zhang W, Krencik R, Deng H, Wang Y, Girton J, Johansen J, Johansen KM. 2005. The JIL-1 kinase interacts with lamin Dm0 and regulates nuclear lamina morphology of Drosophila nurse cells. J Cell Sci 118:5079-5087.
- Berzat AC, Buss JE, Chenette EJ, Weinbaum CA, Shutes A, Der CJ, Minden A, Cox AD. 2005. Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif. J Biol Chem 280:33055-33065.
- Cong X, Nilsen-Hamilton M. 2005. Allosteric aptamers: targeted reversibly attenuated probes. Biochemistry 44:7945-7954.
- Deng H, Zhang W, Bao X, Martin JN, Girton J, Johansen J, Johansen KM. 2005. The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes. Chromosoma 114:173-182.
- Foote MR, Nonnecke BJ, Fowler MA, Miller BL, Beitz DC, Waters WR. 2005. Effects of age and nutrition on expression of CD25, CD44, and L-selectin (CD62L) on T-cells from neonatal calves. J Dairy Sci 88:2718-2729.
- Foote MR, Nonnecke BJ, Waters WR, Palmer MV, Beitz DC, Fowler MA, Miller BL, Johnson TE, Perry HB. 2005. Effects of increased dietary protein and energy on composition and functional capacities of blood mononuclear cells from vaccinated, neonatal calves. Int J Vitam Nutr Res 75:357-368.
- Ji Y, Rath U, Girton J, Johansen KM, Johansen J. 2005. D-Hillarin, a novel W180-domain protein, affects cytokinesis through interaction with the septin family member Pnut. J Neurobiol 64:157-169.
- Lerach S, Zhang W, Deng H, Bao X, Girton J, Johansen J, Johansen KM. 2005. JIL-1 kinase, a member of the male-specific lethal (MSL) complex, is necessary for proper dosage compensation of eye pigmentation in Drosophila. Genesis 43:213-215.
- Qi H, Rath U, Ding Y, Ji Y, Blacketer MJ, Girton J, Johansen J, Johansen KM. 2005. EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila. J Cell Biochem 95:1284-1291.
- Recknor JB, Sakaguchi DS, Mallapragada SK. 2005. Growth and differentiation of astrocytes and neural progenitor cells on micropatterned polymer films. Ann N Y Acad Sci 1049:24-27.
- Sakaguchi DS, SJ VANH, Grozdanic SD, Kwon YH, Kardon RH, Young MJ. 2005. Neural progenitor cell transplants into the developing and mature central nervous system. Ann N Y Acad Sci 1049:118-134.
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Progress 01/01/04 to 12/31/04
Outputs
1) The Buss lab is focusing on defining the role of the C-terminal region in the initial membrane targeting and later plasma membrane subdomain partitioning of HRas. They have made an oncogenic H-Ras protein whose transport on internal membranes is impaired and found that this trapping causes a dramatic reduction in activity. They have also identified a previously unknown step in HRas transport that allows the protein to escape from the cell surface and enter the depths of the cell by a pathway controlled by a protein named Arf6. 2) Girton and his collaborators have demonstrated that chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis. They obtained evidence that the COOH-terminal domain of the nuclear protein Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may contribute structurally to a putative spindle matrix complex. 3) The Becraft lab
identified a promoter element that controls expression of the vp1 gene in maize seeds. This gene controls the decision for seeds to enter dormancy, and understanding how vp1 is regulated will help us understand the developmental regulation of this important process. 4) The Nilsen-Hamilton lab is studying a gene encoding a lipocalin called uterocalin that is expressed in large amounts in tissues that are undergoing involution and during the acute phase response. To test the physiological function of this protein, the lab has created and are currently breeding a transgenic animal to express high levels in particular tissues. The group is also collaborating with others to develop a means of delivering high concentrations of uterocalin to specific tissues from polymers. 5) The Sakaguchi lab are coupling the ability of neural progenitor cells (NPCs) and stem cells, to survive and integrate into host tissue, along with their ability to serve as biological "minipumps", and we have begun to
investigate methods to transduce NPCs and stem cells in an efficient manner using lentiviral vectors. They have successfully transduced a number of different cell types including NPCs (brain and retinal-derived), bone marrow stem cells (MSCs); and rat RGC-5 cells (RGC line) with lentiviral constructs encoding green fluorescent protein (GFP). Moreover, they have successfully expressed foreign genes for brain-derived neurotrophic factor (BDNF) and glia cell line-derived neurotrophic factor (GDNF) in several of these cell populations.
Impacts
One of most important cellular actions is cell division. This is controlled by a complex of internal and external signals. These studies are expected to improve our understanding of how cell division is regulated. In addition, Sakaguchi and Buss's studies hold tremendous potential for the treatment of a variety of degenerative disorders of the nervous system. They are expected to open new uses of stem cells beyond the still-unattained goal of regeneration of specific tissue types, into a broader role as cells to support repair in many degenerative diseases. Nilsen-Hamilton's studies of uterocalin/FREB are expected to lead to a better understanding of the molecular basis of tissue damage in inflammation as occurs in mastitis and Johne's disease.
Publications
- Becraft, P.W. 2004. in: Encyclopedia of Plant and Crop Science.pp. 414 - 417 (Goodman, R.M., Ed.) Marcel Dekker, Inc., New York, NY.
- Cao, X., Li, K., Suh, S.G., Guo, T. and Becraft, P.W. 2005. Molecular analysis of the CRINKLY4 gene family in Arabidopsis thaliana. Planta 220:645-657.
- Determan, A.S., Trewyn, B.G., Lin, V.S., Nilsen-Hamilton, M. and Narasimhan, B. 2004. Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres. J Control Release 100:97-109.
- Grozdanic, S.D., Kwon, Y.H., Sakaguchi, D.S., Kardon, R.H. and Sonea, I.M. 2004. Functional evaluation of retina and optic nerve in the rat model of chronic ocular hypertension. Exp Eye Res 79:75-83.
- Klassen, H., Sakaguchi, D.S. and Young, M.J. 2004. Stem cells and retinal repair. Prog Retin Eye Res 23:149-81.
- Li, M. and Sakaguchi, D.S. 2004. Inhibition of integrin-mediated adhesion and signaling disrupts retinal development. Dev Biol 275:202-14.
- Li, M., Babenko, N.A. and Sakaguchi, D.S. 2004. Inhibition of protein tyrosine kinase activity disrupts early retinal development. Dev Biol 266:209-21.
- Qi, H. et al. 2004. Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila. Mol Biol Cell 15:4854-65.
- Rath, U. et al. 2004. Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor. J Cell Biochem 93:1033-47.
- Recknor, J.B., Recknor, J.C., Sakaguchi, D.S. and Mallapragada, S.K. 2004. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials 25:2753-67.
- Timmermans, M.C., Brutnell, T.P. and Becraft, P.W. 2004. The 46th Annual Maize Genetics Conference. Unlocking the secrets of the maize genome. Plant Physiol 136:2633-40.
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Progress 01/01/03 to 12/31/03
Outputs
The group is analyzing various aspects of cell signaling and gene regulation. The molecules that they are focusing on include enzymes in the MAP kinase pathway (Ras, Raf, MEK), a nuclear-localized protein kinases (JIL-1), a new transcription factor (FREBP), a chromatin proteins (Chromator). In each case, the goal is to understand the molecule's function at a molecular and at a cellular level. The Buss lab is focusing on defining the role of the C-terminal region in the initial membrane targeting and later plasma membrane subdomain partitioning of HRas. They have made an oncogenic H-Ras protein whose transport on internal membranes is impaired and found that this trapping causes a dramatic reduction in activity. Buss and her collaborators are also utilizing a proteomics and bioinformatics approach to identify the proteins present during three stages of retinal development. As possible future treatments for neurodegenerative diseases, the Sakaguchi lab has been
developing experimental strategies using transplantation of neural stem cells and injection of neurotrophic growth factor releasing microspheres into the injured/diseased CNS. They have demonstrated that adult neural stem cells can survive following transplantation into the injured retina. In addition, they found that neurotrophic growth factor releasing microspheres can facilitate the maintenance of visual function in the injured retina. Buss and Sakaguchi are collaborating to engineer neural stem cells to synthesize and secrete neurotrophic growth factors. The genetically engineered stem cells will eventually be tested in a live animal (rat) model of a retinal degenerative disease, glaucoma. Becraft and Nilsen-Hamilton are developing a high-throughput screen for plant receptor ligands by a novel approach using a FRET reporter protein and fusion plant-animal receptors proteins in animal cells. The Girton lab has been studying two genes in Drosophila, JIL-1 and chromator. JIL-1 is a
kinase that acts on histones and whose function is essential for the organization of somatic chromosome packaging. They have successfully generated 45 new mutant alleles of JIL-1, including 12 cold-sensitive and 6 heat sensitive temperature sensitive alleles. They are also studying Chromator with the hypothesis that it is part of a 'spindle matrix' of proteins that guide the assembly and function of the microtubules that make up the visible spindle. They have isolated more than 165 putative mutations to date. The Ambrosio lab has used a molecular genetics approach to define the regulatory roles of CRN (Conserved Region N-terminal) and Conserved Region 2 (CR2) sequences of the D-raf protein. They concluded that both N-terminal and CR2 sub-domains of D-raf play positive roles in the regulation of D-raf by binding to signaling factors that enabled D-raf to participate in EGFR signaling for eggshell production. The Nilsen-Hamilton lab is studying a transcription factor called FREBP that
regulates the expression of a gene that encodes a growth factor and that is highly expressed by the uterus. The lab has evidence that activity of the transcription factor might be regulated by the MAP kinase pathway.
Impacts
One of most important cellular actions is cell division. This is controlled by a complex of internal and external signals. These studies are expected to improve our understanding of how cell division is regulated. In addition, Sakaguchi and Buss's studies hold tremendous potential for the treatment of a variety of degenerative disorders of the nervous system. They are expected to open new uses of stem cells beyond the still-unattained goal of regeneration of specific tissue types, into a broader role as cells to support repair in many degenerative diseases. Nilsen-Hamilton's studies of uterocalin/FREB are expected to lead to a better understanding of the molecular basis of tissue damage in inflammation as occurs in mastitis and Johne's disease.
Publications
- Baker TL, Zheng H, Walker J, Coloff JL, Buss JE. 2003. Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras. J Biol Chem 278:19292-19300
- Buss, J.E. 2003 Membrane Targeting: Methods. Nature Encyclopedia of Life Sciences http://www.els.net/ [doi:10.1038/npg.els.0002615]
- Buss, J.E. et al. 2003. Influence of cellular location on Ras function. In The Handbook of Cell Signaling (Vol. 2) (Bradshaw, R. and Dennis, E., eds.), pp. 675-679, Elsevier Science
- Grozdanic S., Betts D.M., Allbaugh R.A., Sakaguchi D.S., Kwon Y.H., Kardon R.H., Sonea I.M. 2003. Characterization of the pupil light reflex, electroretinogram and tonometric parameters in healthy mouse eyes. Curr Eye Res 26:371-378.
- Grozdanic S.D., Betts D.M., Sakaguchi D.S., Allbaugh R.A., Kwon Y.H., Kardon R.H. 2003. Laser-induced mouse model of chronic ocular hypertension. Invest Ophthalmol Vis Sci 44:4337-4346.
- Grozdanic S.D., Betts D.M., Sakaguchi D.S., Kwon Y.H., Kardon R.H., Sonea I.M. 2003. Temporary elevation of the intraocular pressure by cauterization of vortex and episcleral veins in rats causes functional deficits in the retina and optic nerve. 2003. Exp Eye Res 77:27-33.
- Grozdanic S.D., Sakaguchi D.S., Kwon Y.H., Kardon R.H., Sonea I.M. 2003. Functional characterization of retina and optic nerve after acute ocular ischemia in rats. Invest Ophthalmol Vis Sci 44:2597-2605.
- West, H., Greenlee M., Uemura E., Carpenter S.L., Doyle R.T., Buss J.E. 2003. Glucose uptake in PC12 cells: GLUT3 vesicle trafficking and fusion as revealed with a novel GLUT3-GFP fusion protein. J Neurosci Res 73:518-525.
- Kwon, S.-H., Kim, S.H., Chung, H.-M., Girton, J.R., and Jeon, S.-H. 2003. The Drosophila pleiohomeotic mutation enhances the Polycomblike and Polycomb mutant phenotypes during embryogenesis and in the adult. International Journal of Developmental Biology 47:385-395.
- Nilsen-Hamilton M., Liu Q., Ryon J., Bendickson L., Lepont P., Chang Q. 2003. Tissue involution and the acute phase response. Ann N Y Acad Sci 995:94-108.
- Nilsen-Hamilton M., Werb Z., and Keshet E., editors. 2003. Tissue Remodeling, Annals of the New Academy of Sciences, Vol. 995, 215pp, New York Academy of Sciences, N.Y.
- Sakaguchi D.S., Van Hoffelen S.J., Young M.J. 2003. Differentiation and morphological integration of neural progenitor cells transplanted into the developing mammalian eye. Ann N Y Acad Sci 995:127-139.
- Zhang, W., Ye J., Girton, J., Johansen, J., Johansen, K.M. 2003 Genetic and phenotypic analyses of alleles of the Drosophila chromosomal JIL-1 Kinase reveals a functional requirement at multiple developmental stages. Genetics 165:1342-1354. JIL-1
- Zhang, W., Wang, Y., Long, J., Girton, J., Johanen, J., and Johansen, K.M. 2003. A developmentally regulated splice variant from the complex lola lous encoding multiple different zinc finger domainproteins interacts with the chromosomal kinase JIL-1. The Journal of Biological Chemistry, 278:11696-11704.
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Progress 01/01/02 to 12/31/02
Outputs
Studies were performed of cellular signal transduction components including cellular protein kinases, phosphatases, G-proteins and transcription factors. These studies focused on how the enzymes were regulated and identifying the cellular context in which the enzymes function. Integrated cellular functions were also studied to understand how cells interact with their environment during tissue remodeling. The Becraft lab has completed a molecular analysis of the CR4 family of receptor kinases in Arabidopsis and has begun to develop an assay for plant receptor/ligand interactions using a mammalian reporter system. They have submitted over 20,000 cDNAs for sequencing for the EST project of the "Functional genomics of maize endosperm development" consortium. The Norris lab studies the role of inositol polyphosphate 4-phosphatase (IP4P) in the regulation of PI 3-Kinase (PI3K) mediated signaling. PI3K activation results in the accumulation of two second messengers, one of
which is a substrate for IP4P. The cDNA encoding the C. elegans IP4P has recently been cloned and characterized. Two mutant alleles have also been isolated. A genetic analysis was completed by the Girton lab of JIL-1, a gene in Drosophila whose product is a kinase which acts on Histone H3, and appears to function to regulate chromatin structure and gene regulation. A mutagenesis experiment is currently in progress to recover new conditional mutations of JIL-1. The Ambrosio lab has found that Conserved Region 2 of D-raf interacts with arl1. With the EGF receptor Arl1 establishes dorsal-ventral pattern of the egg shell and embryo. Arl determines cell identity at the anterior and posterior poles of the embryo under the control of torso, a tyrosine kinase (PTK) receptor. Since D-raf also participates in these signaling pathways a link has been established between D-raf, PTK signaling pathways and arl1 function in Drosophila. The Buss lab has shown that palmitate is more potent than
isoprenoid for membrane attachment and is fully capable of supporting Ras biological activity by itself. Moreover, there is a profound acceleration in the cycling of palmitate in oncogenic forms of the protein. This finding is notable because it suggests that the dynamic effects of palmitoylation may be exaggerated in oncogenic proteins. As a strategy for treating neurodegenerative diseases, the Sakaguchi lab is developing procedures for the transplanting neural stem cells in the CNS. The goal is to understand factors and conditions that influence the control of development in neural stem cells, with the hope of developing procedures to repair the damaged nervous system, including the retina, of human patients. They have demonstrated that the age of the host environment strongly influences the ability of neural stem/progenitor cells to integrate structurally into the eye. The Nilsen-Hamilton lab is purifying the FREBP, a transcription factor that activates the mrp/plf genes that
encode growth and angiogenesis factors. Expression of the mrp/plf genes was found in the intervertebral discs of the developing fetal spine. A mathematical model was developed in collaboration with Levine to describe angiogenesis.
Impacts
Understanding the molecular basis of cellular function and how it is regulated will provide a rational means of regulating cellular growth and differentiation for more healthy animals and plants. The use of stem cell transplants holds tremendous potential for the treatment of a variety of degenerative disorders of the nervous system.
Publications
- Becraft PW. 2002. Receptor kinase signaling in plant development. Annu. Rev. Cell Devel. Biol. 18:163-192.
- West Greenlee MH, MC Wilson and DS Sakaguchi. 2002. Expression of SNAP-25 during mammalian retinal development: Thinking outside the synapse. Seminars Cell Dev. Biol. 13:99-106.
- Becraft PW, K Li, N Dey and YT Asuncion-Crabb. 2002. The maize dek1gene functions in embryonic pattern formation and in cell fate specification. Development 129:5217-5225.
- Burke DH, NDS Ozerova and M Nilsen-Hamilton M. 2002. Allosteric Hammerhead Ribozyme TRAPs, Biochemistry 41:6588-6594.
- Grozdanic S, DS Sakaguchi YH Kwon RH Kardon and IM Sonea. 2002. Characterization of the pupil light reflex, electroretinogram and tonometric parameters in healthy rat eyes. Current Eye Research. 25:69-78.
- Levine HA, S Pamuk, BD Sleeman and M Nilsen-Hamilton. 2002. Mathematical modeling of tumor angiogenesis and the action of angiostatin as a protease inhibitor. J. Theoretical Medicine 4:133-145.
- Li M and DS Sakaguchi. 2002. Expression patterns of focal adhesion proteins in the developing retina. Developmental Dynamics. 225:544-553.
- Prapong T, J Buss, WH Hsu, P Heine, HW Greenlee and E Uemura. 2002. Amyloid beta-peptide decreases neuronal glucose uptake despite causing increase in GLUT3 mRNA transcription and GLUT 3 translocation to the plasma membrane. Exper. Neurol. 174:253-258.
- Ryon J, L Bendickson and M Nilsen-Hamilton. 2002. High Expression in Involuting Reproductive Tissues of Uterocalin, a Lipocalin and Acute Phase Protein, Biochem J. 367:271-277.
- Van Hoffelen SJ, MJ Young, M Shatos and DS Sakaguchi. 2003. Incorporation of murine brain progenitor cells into the developing mammalian retina. Investigative Ophthal. Visual Sci. 44:426-434.
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