(Mentor: Dr. Jonathan Jarvik)
Cell-based Assay for Microtubule Destabilizing Drugs Using an NIH 3T3 Cell Line with a GFP Tag in the Gene for a-tubulin1
CD-tagging is a novel approach to simultaneously mark genes and their products using a retroviral vector encoded with GFP. One cell line produced by this method expresses a GFP tagged a-tubulin. The functionality of this tagged protein can be determined by observing a known behavior induced by a drug, such as nocodazole, a well-known microtubule-binding drug. Many anticancer agents disrupt the mitotic spindle and cytoskeleton by binding the b-subunit, causing the cell to reversibly lose form and function. The goal of this project was to determine if GFP-tagged a-tubulin cells could be used in functional assays for new antitumor microtubule destabilizing drugs. GFP-tagged a-tubulin cells were treated with nocodazole and visualized using spinning confocal microscopy. Depolymerization of microtubules occurred rapidly while polymerization of microtubules after the drug was removed occurred more slowly. Depolymerization of all microtubules except a few persistent ones was seen within ten minutes after drug administration (10 mg/ml). We conclude that the cells expressing GFP-tagged a-tubulin have promise for use in cell-based assays for microtubule destabilizing drugs.
(Mentor: Dr. Alison Barth)
Mapping Plasticity and Gene Expression in the Cortex of fosGFP Transgenic Mice
Behavioral plasticity can be induced through manipulation of sensory experience, e.g. by removal of whiskers. In order to gain a greater understanding of neocortical plasticity within the barrel cortex, we plan to use a strain of transgenic mice carrying a foreign gene composed of the gene for green fluorescent protein (GFP) fused with the immediate-early gene c-fos to track increased cellular activation following whisker deprivation. First, it is necessary to create and identify a strain of mice which are positive for the fosGFP transgene where fosGFP expression mirrors endogenous fos expression. To accomplish this, immunohistochemistry was employed to allow for the specific labeling of the fos protein, which when overlaid or merged with the natural fluorescence of GFP will provide information regarding their relative cellular loci. If the transgene is regulated appropriately, we expect that the fos protein will be present in the same nuclei as the fosGFP fusion protein. It is expected that the results will validate previous studies utilizing the c-fos gene as a marker of neuronal activation. After conclusive identification of different fosGFP lines, those with the best cortical expression will be utilized in a study inducing expression in the cortical barrel field. The fos protein is known to have a half life of two hours but the novel fosGFP fusion protein has yet to be characterized in this manner. To determine the half life of this fusion protein in the animal, an in vivo time course was used. The transgenic mice were plucked such that only one previously designated and consistent facial vibrissae remained on the right side of the muzzle. These mice were then exposed to a novel environment to promote whisking activity for a 24 hour time period. Following this 24 hour period, the remaining (D1) whisker was removed and the mouse returned to the enriched environment for a period of zero, two, four, six, or sixteen hours before the animal was sacrificed. This period of time following removal of the one remaining whisker represented the end of vibrissae stimulation. The brains were then equilibrated in sucrose and sliced on a cryotome in 50 micron sections. Sections were treated with anti-cfos antibody and then labeled with Cy3, a red secondary antibody. The sections were examined under both rhodamine and GFP filters on a fluorescent microscope to locate the barrel corresponding to the stimulated facial vibrissae. The number of fos positive and GFP positive cells were tabulated for the stimulated barrel and the adjacent barrel. Results confirmed that the fos protein has a half life of two hours, and indicated that the half life of the novel fosGFP protein is longer. Future experimentation on these animals will include electrophysiological recording from green (recently activated) cells and non-green (control) cells. Results from these pending studies will hopefully lead to an increased understanding of the neuronal bases for learning and behavior.
(Mentor: Dr. Nathan Urban)
Characterization of Odor Evoked Activity in Olfactory Bulbs using a Fos-GFP Transgenic Mouse
Odor receptors on olfactory sensory neurons (OSNS) bind odor molecules and this binding yields an action potential firing. Axons of the OSNS travel to the olfactory bulbs where they synapse on mitral cells in ~ 2000 glomeruli on the surface of the bulb. There, the glomeruli receive, sort, and pass on this information. It is the goal of this project to characterize the patterns of glomeruli that are activated by various kinds of olfactory experiences. We will use a transgenic mouse in which green fluorescence protein, GFP, expression is linked to the immediate early gene cFOS. GFP expression will then serve as an indicator of neural activity. Olfactory systems of transgenic mice will be stimulated by both odorant exposure and odorant exposure following naris occlusion. The olfactory bulbs will be removed and sectioned for microscopy. Activated glomeruli will be located through fluorescence imaging and, once identified, the degree of activation will be quantified as a function of odorant concentration and time following odor presentation. We hypothesize that increasing odor concentration will yield broader activity across glomeruli and that an optimal time peak for glomerular activity will be observed.
(Mentor: Dr. John Woolford)
Cell Cycle Effects of Mutations in Ribosome Assembly
Yeast mutants have helped us to start understanding the function of certain genes in ribosome assembly and the cell cycle. There are several known mutants that are known to affect ribosome assembly but are not known to affect the cell cycle. The goal of this research is to better understand the role that ribosome assembly mutants have on the cell cycle. In order to do this, I used microscopy to analyze different phenotypes in mutants whose functions are not known in the cell cycle.
(Mentor: Dr. Amy Csink)
Cis-inactivation by heterochromatin of the bwD allele in Drosophila melanogaster
In most animal cells, approximately 90% of the chromatin is transcriptionally inactive. Mechanisms that turn a gene off can be just as vital to the survival of a cell or multicellular organism as mechanisms that turn on a gene. Heterochromatin often plays an important role in the silencing of genes. If a gene is close enough to heterochromatin on the same chromosome and is silenced, it is called cis-inactivation. Trans-inactivation occurs when a silenced gene is not on the same chromosome as the heterochromatin but is in the same area during interphase. Previous work in the Csink lab has been successful in studying trans-inactivation in Drosophila melanogaster using bwD, an allele of the brown eye color gene (bw), which contains a 1.6 megabase heterochromatic insert. The goal of the current research is to use bwD to study cis-inactivation of a reporter gene in a transposon which has been inserted 15 kb away from the heterochromatin in the bwD allele. The reporter gene contains a version of the white gene that allows eye pigments to be transported. At its starting location the white gene is not silenced by bwD heterochromatin in cis or trans. Males with the bwD allele, the transposon containing the reporter gene, and a transposase were crossed to females. Previous experiments in the lab found that the transposon inserted in the site used did not jump along the same chromosome, but instead the transposase caused flanking deletions. We speculated that if such a flanking deletion brought the reporter gene closer to the bwD heterochromatic insertion it would result in the silencing (partial or total) of the reporter gene and hence a change in the eye color. A total of 4,706 flies were screened, and mutant progeny that were variegated or white eyed were collected. Of the 4,706 flies screened, 1.3 % of the progeny were variegated (60 flies) and 3.5 % of the progeny were white eyed (167 flies). The white eyed flies could result from either total silencing of the reporter gene or the removal of the reporter gene in the event that the transposon left the chromosome. Preliminary PCR analyses are being performed on each of the mutant fly lines to determine if the transposon is still present and if there are simple flanking deletions between bwD and the reporter gene. Lines that have flanking deletions resulting in some degree of cis-inactivation will be maintained for further study. The data from the flies will be analyzed to see if there is a correlation between the distance from the heterochromatin and the degree of silencing. The results will lead to further study to compare both quantitatively and qualitatively cis- and trans-inactivation.
(Mentor: Dr. Jonathan Minden)
Triggered apoptosis in wingless mutant Drosophila embryos: Determination of the signal sequence of cell death
Just as in the course of development of many animals, apoptosis, or programmed cell death, occurs naturally in Drosophila melanogaster. In circumstances that benefit this organism, excess cells that occur naturally are eliminated. Cell death also occurs as a response to defects in embryonic patterning or morphogenisis. Elimination of unnecessary cells occurs in a regulated manner, which suggests a gene-directed process. Wingless is an essential gene for patterning and segmentation in Drosophila. Mutation of the wingless gene in Drosophila embryos, leads to an increase in cell death, which is initiated by the cell death gene reaper (rpr) gene. However, the sequence of events that occur between wingless misfunction and expression of rpr are not known. The goal of this project is to identify proteins upstream of reaper that are involved in this sequence. In order to do this, temperature sensitive wingless embryos were compared to wild-type embryos using 2-dimensional Difference Gel Electrophoresis (DIGE). DIGE is a technique in which two samples of protein are labeled differently and run on the same gel. However, because a mutation on the wingless gene may also trigger other processes, it was important that the embryos were analyzed immediately after the start of cell death. This, in turn, will give the correct proteomic differences between wildtype and wingless mutant embryos. To do this, several embryos from both wildtype and wingless flies were injected pre-cellularization with acridine orange, a fluorescent cell death indicator. Next, 3D Time-Lapsed Microscopy was used to photograph the specified embryos by both transmitted light and fluorescence microscopy in small increments over a given period of time. From the fluorescence microscopy we found that apoptosis begins earlier in wingless embryos than in wildtype embryos. Thereafter, in comparison to wildtype, wingless embryos tend to have an average of 45% more apoptosis in each stage of development. We correlated the appearance of dead cells with a specified timepoint after a certain stage of embryonic development (formation of the cephalic furrow), and were therefore able to determine the precise time of the onset of cell death. This gave us a timepoint for when the two types of embryos should be collected, stored and prepared for DIGE. Protein extracts from the embryos were labeled either with cy3 or cy5 and run on reciprocal 2-dimentional gels. Difference proteins were observed in comparison between wingless and wildtype. These proteins will be digested and Mass Spectrometry will be used to identify them. Hopefully, this will give us a lead into the upstream signaling sequence of Drosophila embryonic apoptosis.
|Nikaury Rivera Antongiorgi
(Mentor: Dr. Javier Lopez)
Quantitative Analysis of Lariat Intermediates for Alternative Splicing
Genes in eukaryotes are often interrupted by intervening sequences (introns) that must be removed during gene expression. RNA splicing is the process by which introns are removed and the flanking sequences (exons) are joined together. Alternative splicing of pre-mRNA is an important mechanism for generation of structural and functional protein diversity. Alternative splicing is frequently regulated developmentally, and mutations can affect splicing of specific introns to cause various diseases. Elucidation of splicing mechanisms will help in finding new treatments for genetic diseases and lead to a better understanding of genetic information and gene expression. Several methods are being developed for analysis and comparison of alternative splicing across the entire genome. One of these methods is based on analysis of the lariat intermediates from the first step of splicing, which has several advantages over analysis of the final mRNA products. The goal of this project was to test the suitability of lariat analysis for quantitation of alternative splicing events in different systems. For this purpose, a recently developed reverse transcription/polymerase chain reaction assay was used to detect the lariat intermediates corresponding to well-characterized splicing events in three different systems. The first system involved exon mI in the Ubx gene of Drosophila. Exon mI is inappropriately excluded from Ubx mRNAs of mutants where exon mII is deleted. It has been proposed that this is a result of resplicing exon mI after it has been joined to the upstream exon. The unique advantage of lariat analysis was tested for distinguishing between exon skipping and resplicing mechanisms of exon exclusion. Lariat analysis confirmed that exon mI is excluded by exon resplicing rather than exon skipping. The second system was the human E2F4 mRNA, which is expressed at high levels but has several odd small introns that have repeated 3' splice site signals and lack consensus branchpoints. Lariat analysis was used to attempt to identify the branchpoint for E2F4 intron 7. A computer algorithm predicted several stable structures that may be interfering with lariat amplification. It was tentatively concluded that the branchpoint for intron 7 is approximately 50 nucleotides upstream from the 3' splice site. The third system was the RPS14B mRNA of Saccharomyces cerevisiae, whose splicing is repressed by the RPS14A gene product. RPS14B lariat levels are normally very low but should increase in a rps14a mutant strain. Lariat analysis was attempted to quantitate repression of RPS14B splicing by RPS14A protein. Lariat analysis showed the expected de-repression of RPS14B in a rps14a deletion mutant. It was also found that RPS14A may contain an alternative 3' splice site within exon 2. In future research, lariat analysis will be a valuable tool for analysis of splicing regulation in vivo.
(Mentor: Dr. Charles Ettensohn)
Analysis of Nuclear Lytechnius variegatus Beta-catenin Localization in Early Sea Urchin Embryos
Beta-catenin is a co-transcriptional regulator, often activated by the Wnt pathway, that is known to play an important role in the specification of micromeres in the sea urchin embryo. It is known that the presence of this protein in the nuclei of vegetal cells is required for normal endoderm and mesoderm development. Beta-catenin appears to be involved in cell fate specification signaling in all deuterostomes studied to date The similarities between echinoderm and chordate beta-catenin distribution suggest a conservation of molecular networks regulating axial patterning events.
Previous work in our lab using GFP-tagged beta-catenin from Xenopus, has shown that this protein degrades at a faster rate in animal cells than in vegetal cells of sea urchin embryos. Recently, beta-catenin from Lytechinus variegates has been sequenced. It is the purpose of this project to show that L. variegates beta-catenin degrades from animal pole cells in the same manner as Xenopus beta-catenin. A plasmid vector containing a GFP-tagged L. variegates beta-catenin sequence (received from Dr. David McClay) was used in a T3 in vitro transcription reaction to obtain mRNA encoding GFP-tagged L. variegates beta-catenin. This mRNA was injected into fertilized sea urchin eggs. A lack of green fluorescence after the first few cleavages indicated that the RNA was not being expressed. Since a lack of expression might have been due to the vector, primers were designed, and PCR was used to clone the L. variegates sequence into a vector that has previously worked in other experiments involving expression. After mRNA was made in the same manner (using a SP6 in vitro transcription reaction), fertilized embryos were again injected. The injected embryos showed slight fluorescence, and it was apparent that the protein had appropriately localized in the nuclei of the cells. Some grainy cytoplasmic staining was also apparent. Thus far, the L. variegates beta-catenin has localized in the same manner as the Xenopus beta-catenin.
Further work, following more injections, could include experiments with emetine, which will block further translation after the initial accumulation of protein. The level of fluorescent intensity of cells along the animal-vegetal axis will then be measured and analyzed using time lapse microscopy and image processing software. The half-life of the protein in the two poles of the embryo can then be compared.
(Mentor: Dr. Robert F. Murphy)
Computational Models of Drug Effects on Protein Location
Determining the subcellular location pattern of proteins is crucial to understanding their function. A numeric representation for protein location has been previously described which allows the creation of a systematics of the locations of all proteins in a proteome by way of hierarchically clustering groups of proteins that have similar localization. However, some proteins in a given location group may respond differently to environmental or pharmacological conditions and this provides a way to further subdivide the hierarchy. Therefore we have explored here methods to quantify such responses. We used four 3T3 cell clones that each express a different GFP fusion protein. These clones were created by CD-tagging, a process that puts a specific molecular tag on random genes, their transcription products and protein products. We collected images of these proteins in the presence and absence of four different drugs. We collected 3D images with a spinning disk confocal microscope for each cell line for each drug and control images for condition. We then compared a set of numerical features (Subcellular Location features) to describe each image. It has been shown that all major subcellular locations can be distinguished based on these features. We compared the images using the SimEC (Statistical Imaging Experimental Comparator) system that provides rigorous statistical comparison of sets of images.
(Mentor: Dr. Adam Linstedt)
Evidence that the matrix, a putative template for Golgi biogenesis, is extensively disassembled and dispersed during mitotic Golgi disassembly
During mitosis, the mammalian Golgi disassembles into numerous vesicles and larger membrane structures referred to as clusters or remnants. Following mitosis, the vesicles and clusters reassemble to form the Golgi structure in the daughter cells. Previous research has suggested that certain Golgi structural proteins such as GM130, giantin, and the GRASPs are enriched in the clusters and serve as a stable template for reassembly of the Golgi in the daughter cells. The purpose of this study was to determine if Golgi "matrix" proteins are preferentially sorted into the cluster/remnant structures. Due to their relatively large size, individual clusters may be resolved by light microscopy. Vesicles, however, are too small to be differentiated and are seen as a haze. It was necessary, therefore, to devise a method by which an accurate comparison of protein content in clusters vs. haze could be made. Images of mitotic NRK cells stained by indirect immunofluorescence were acquired using a deconvolution microscope, and a 3D-computational analysis was performed to determine if the "matrix" proteins were enriched in the mitotic clusters in comparison with other "non-matrix" Golgi resident proteins. The percent fluorescence found in clusters versus haze, number of clusters per cell, and average size of the clusters was determined. For both "matrix" and "non-matrix" Golgi proteins, only a small percentage of total fluorescence was found to be in the clusters, and no significant difference was seen between the average number and size of the clusters. A visual analysis of the fluorescent images also suggested that the Golgi proteins are not sorted during mitosis. In cells stained with antibodies generated against "matrix" and "non-matrix" Golgi markers, a similar pattern was observed for both proteins. Analysis of overlayed images of two different markers revealed that the Golgi proteins localize directly adjacent to one another in the clusters. This indicates that the proteins are in the same clusters but not sharing the same membrane space. The results from this study suggest that, although Golgi "matrix" proteins may mediate Golgi formation in the daughter cells, the mechanism by which this is accomplished does not involve a stable template comprised of "matrix" proteins persisting in the clusters/remnants.