Robert Fleming - US grants

9418678 Fleming One mutation in Drosophila melanogaster which exhibits neuronal hyperplasia with concomitant reduction in hypodermal structures has been named Notch. Serrate, another gene mutation, appears to play an important developmental role in interacting with the Notch gene product to mediate cell- to- cell signaling during the development of tissues other than the early embryonic neuroblast, where the product of another gene, Delta, seems to interact with Notch. Since Ser-null alleles cause larval death, it has not been possible to identify which tissues require Ser for viability or the possible role of Ser post-embryonically. New approaches will be utilized to answer the questions: (1.) What is the essential role played by Serrate during development? (2.) Where and when is the Serrate protein expressed during development? (3.) What is the nature of the class of dominant Serrate mutations that interact strongly with null Delta alleles? %%% Experiments are proposed to investigate the function of the Serrate gene product in the fruit fly, Drosophila melanogaster. A number of genetic loci have been isolated on the basis of a common mutant phenotype and studies have suggested that most of these genes products are interactive in a manner necessary for the differentiation of neuro-ectoderm. The function of one of them, Serrate, will be examined in relation to its function in embryonic development and in the manner in which it interacts with products of other related gene loci. ***

Fleming, Robert J.. 9727951 During the development of multicellular organisms, single cellular receptors are often used at multiple developmental periods. The Notch gene family represents a highly conserved group of genes whose functions are required in organisms as diverse as nematodes and humans. Notch and Notch-related gene products function as cell membrane receptors for intercellular signaling events to coordinate cell fate decisions in a variety of tissues during development. Notch activity is regulated by membrane-bound ligands which, in Drosophila, are represented by the products of the genes Serrate (SER) and Delta (DL). SER-like and DL-like molecules have also been found to be conserved in the same species where Notch family genes are found. These ligands demonstrate distinct temporal and spatial expression patterns suggesting that they each regulate NOTCH in particular processes. Interestingly, both SER and DL can act to initiate NOTCH signaling and appear to have equivalent capabilities during some processes yet function distinctly in others. Because SER and DL can elicit distinct responses from the single NOTCH receptor and since NOTCH is central to many cell fate decisions, these studies will investigate the mechanisms by which specificity is generated by each ligand upon association with NOTCH. The proposed experimentation will examine the roles of specific protein domains within SER and DL that contribute to the ability of that ligand to activate NOTCH in different cellular environments. Mechanisms of ligand function will be determined by constructing mutations of extracellular or intracellular (IC) domains of each ligand and expressing the mutant molecules in vivo. By analyzing the properties of these chimeric molecules relative to normal SER and DL, specific functional properties produced by individual protein domains will be determined. Initially, studies will focus on possible parallel mechanisms between NOTCH ligands and TGF(-like molecules that require IC domains f or proper extracellular function. Preliminary studies in these areas suggest that SER molecules function cooperatively and may activate NOTCH as a dimer or other aggregated form during some aspects of NOTCH signaling. These findings suggest other areas of experimentation including molecular examination for SER cleavage products and functional interactions between extracellular region mutations and the IC domain. Other areas of investigation include the characterization of a unique cysteine-rich domain found only within the SER ligand and not in DL. The properties of this domain will be characterized by deleting it from SER and/or inserting it into DL and assessing the ability of these modified ligands to interact with and/or activate the NOTCH receptor in specific cellular environments. Taken together, these studies are expected to expand the understanding of NOTCH signal regulation and further clarify mechanisms by which receptors in general may serve to send multiple, discrete signals upon activation by theft identified ligands.

0234751
Fleming

In eukaryotic cells, cytoplasmic and nuclear compartments are separated by the nuclear envelope. This double membrane structure contains numerous nuclear pores that regulate the flow of molecular information into and out of the nucleus. Proper functioning of this transport process is essential for normal cellular metabolic regulation as well as the transmission of developmental signals from the extracellular and cytoplasmic compartments to the nucleus so that gene transcription can be regulated and modified during cellular differentiation processes.
Many specific transporter molecules have been identified in this process. In particular, Dr. Fleming will focus on the importin a (imp a) genes act as adapter proteins for cargo molecules containing classical nuclear localization signals. The imp a gene family contains three distinct members (clades): imp a1, a2 and a3. Metazoan animals generally have representatives of each of the three clades. The evolutionary conservation of all three clades from Drosophila through human suggests that each clade is constrained in its ability to diverge from or converge on the others, suggesting that each clade has evolved specialized functions. He has chosen to examine the imp a gene family in Drosophila since there are only single genes representing each of the clades, thus allowing us to assess any specific functions of individual clade members.
The merit of these studies resides in the definitive demonstration of unique roles for specific imp a genes in nuclear transport and perhaps roles for these genes in other cellular processes. The elucidation of these functions is critical for the understanding of cellular signaling and homeostasis. It is expected that these roles will be conserved in other metazoan animals having representative members of the different imp a clades. His studies will reveal evolutionary conservation of specific functions by comparisons with other species.
Additionally, the experimentation within this proposal will provide undergraduate students with direct exposure to current biological methodologies while demonstrating the extent to which conclusions can be drawn from available data. This exposure will allow students to design additional experiments based on their findings and explore the true nature of scientific thought and experimentation. It is further anticipated that students will experience the opportunity to present their findings at local and national meetings and also in publications to experience the totality of the scientific community and the interactions fostered by it.

The Notch signaling system is a cell-to-cell communication mechanism that is found in virtually all multicellular organisms and allows developing cells to acquire their specialized cellular types (neuronal, muscular, digestive, reproductive, etc.). This system has many components, two of which are the main focus of this proposal. These are the Notch signal receptor molecule and the Serrate ligand that is capable of interacting with and affecting Notch receptor activity. The Serrate ligand can activate the receptor when these molecules are expressed on adjacent cells leading to the formation of different cell types. Serrate can also inhibit the receptor when these molecules are co-expressed on the same cell, preventing such cells from receiving a Notch signal and forming distinctive cellular types. This proposal investigates the specific regions and properties of the Serrate ligand that are responsible for inhibiting the Notch receptor hence investigating a crucial mechanistic aspect for controlling cellular specialization. The work utilizes targeted molecular-genetic mutation of the Serrate molecule with subsequent expression of these mutated forms in the fruit fly to investigate their effects on Notch signaling. The outcomes are expected to fully define and characterize the regions of Serrate that confer the inhibitory property of the ligand onto the Notch receptor and potentially define regions essential for Notch activation. The evolutionarily conserved nature of Notch system components allows for very broad applicability of the findings of this study to the homologous molecules in vertebrates. Further, this study will extend the understanding of cellular differentiation control in multicellular animals including stem cell differentiation mechanisms. The study will be conducted by undergraduate researchers providing these students with a strong background in molecular and genetic tools for subsequent professional training and development.