Graduate Summer Colloquium 2009:

Scott Topper

“Downstream, Upstream: Genomic Approaches to Understanding the Environmental Stress Response”

Yeast respond to significant changes in their environment by activating massive alterations in their transcriptome. While some of these changes are specific to particular stresses, a core set of 900 gene transcripts are coordinately induced or repressed in response to many diverse stresses that have very different physiological consequences for the cell. Downstream: While the transcriptional response to stress is very well characterized, there is sparse evidence to describe the global changes at the protein level. Through a collaboration with Joshua Coon and Violet Lee (UW-Madison), we are using a semi-quantitative proteomics method to identify changes in protein level over time, and working to correlate those protein level changes to gene expression changes. I will present early data on the protein-level changes in response to osmotic shock, and describe our initial observations on the relation between translation to transcription in stress-induced changes.

Upstream: While much is known about the pathways which activate gene expression in response to particular stresses, the full genetic network governing gene activation is only slowly coming together. I will also describe a simple, fluorescence-based, flow-cytometric, genetic screen we are developing to identify regulators of the ESR, and to flesh out the general and stress-specific signaling networks regulating the response.

Eric Domyan

“Signaling through BMPR1A and BMPR1B regulates D-V patterning of the foregut endoderm and early budding events of the lung”

The mammalian foregut endoderm gives rise to the dorsally-located esophagus and stomach, and the ventrally-located trachea and lungs. Proper patterning and morphogenesis of the common foregut tube and its derived organs is essential for viability of the organism. Previous studies suggest that BMP signaling is important for early stages of respiratory development, although its precise role remains unclear. Here we examine the role of Bmp Type I receptors BMPR1A and BMPR1B in this process. Mice in which Bmpr1a and Bmpr1b have been removed from the ventral endoderm displayed tracheal agenesis, ectopic primary bronchi, and dialated lung buds. Molecularly, we observed that loss of Bmpr1a and Bmpr1b caused a reduction of ventral endoderm marker NKX2.1 and an expansion of dorsal markers in the early anterior foregut, as well as in parts of the early lung. Together, our data suggest that signaling through BMPR1A and BMPR1B regulates D-V patterning of the foregut endoderm and early budding events of the primitive lung.

Jessica Shivas

“Dynamin participates in the maintenance of anterior embryonic polarity”

Cell polarity is crucial for the generation of cell diversity in a multicellular organism. During the first cell cycle, the C. elegans embryo polarizes along its anterior-posterior axis through the formation of two separate cortical domains consisting of the anterior and posterior PAR proteins. This process occurs in two distinct phases: establishment and maintenance. Much has been learned about the role of acto-myosin-associated mechanisms required for establishing these cortical domains, but the factors required to maintain polarity cues in particular membrane domains during development remain elusive. Current models suggest that PAR-dependent membrane recycling may play a key role in polarity. However, the links between the PAR proteins, endocytic machinery and the actin cytoskeleton during development are unclear. Dynamin is a large GTPase that plays key roles in both endocytosis and actin dynamics and, therefore, a candidate that could connect these events to cell polarity pathways. We have found that the C. elegans dynamin, DYN-1, is required to maintain the previously established PAR asymmetry. Additionally, we found an enrichment of DYN-1 in the anterior cortex, as well as an enrichment dynamin-dependent endocytosis from the anterior membrane during the polarity maintenance phase. Our results suggest a new role for DYN-1 in regulating asymmetric endocytosis, and subsequently, the maintenance of PAR asymmetry.

Mary Kate Bonner

“Metaphase spindle proteome reveals potential furrow initiation factors ”

Cytokinesis is an important and fundamental process in the development of all organisms. The factors that establish the cleavage furrow have remained mysterious and have eluded many for over 130 years. In order to identify factors required for early steps in cytokinesis, mitotic spindles from synchronized Chinese Hamster Ovary (CHO) cells were isolated. Proteins enriched from isolated metaphase-enriched spindles were identified by multidimensional protein identification technology (MudPIT) in collaboration with the Yates Lab at Scripps. A comparative genomics analysis between the spindle and the midbody proteome (Skop et al, 2004) identified potential candidates.

Preliminary results from multiple MudPIT data sets identified ~2000 proteins with two or more peptide hits. We compiled the spindle proteome by averaging four MudPIT data sets. We prioritized our list of candidates by identifying proteins specific to the metaphase proteome, using Babelomics (http://babelomics.bioinfo.cipf.es/) and Microsoft Access. We are particularly interested in membrane-cytoskeleton remodeling proteins, as these factors are likely involved in establishing and regulating the acto-myosin contractile ring. We are currently screening several homologs of the identified mammalian candidates in C. elegans using RNAi. Potential candidates include factors that function in furrow formation. We are further characterizing candidates using in vivo microscopy, genetics and cell biological techniques.

JJ Chritton

“Combinatorial control of mRNA expression by PUF proteins”

PUF proteins are a ubiquitous family of RNA binding proteins. PUF proteins down-regulate protein expression by binding to mRNAs and repressing them. Repression is initiated by the recruitment of proteins that destabilize the message and promote decay. However, PUF proteins do not depend solely on this mechanism for repression.

I have developed a cell-free translation system derived from yeast that recapitulates PUF repression in vitro and enables careful dissection of the mechanisms behind repression. Using this system, I have analyzed several putative targets of Puf5p in yeast and found that Puf5p regulation varies both in strength and mechanism based on the target mRNA. I have also identified elements outside of the Puf5p binding element that enhance Puf5p repression.

Allison Lynch

“MAGI-1 modulates the C. elegans cadherin-catenin complex during morphogenesis”

In multicellular organisms, cell-cell junctions are involved in all aspects of tissue morphogenesis, including regulation of cell shape and movement. To establish junctions, cytoplasmic proteins that interact with the actin cytoskeleton must stabilize adhesion molecules. For example, catenins connect cadherins to the cytoskeleton and mutations in catenin proteins significantly impact morphogenesis. In continuing efforts to identify regulators of the cadherin-catenin complex, we conducted a genome-wide RNAi screen and uncovered MAGI-1. We have shown C. elegans MAGI-1 genetically interacts with hmp-1/?-catenin and hmp-2/?-catenin, suggesting MAGI-1 may modulate cadherin-catenin function during morphogenesis. MAGI-1 localizes at the C. elegans apical junction, but is surprisingly basal to the cadherin-catenin complex. We are currently examining how MAGI-1 gets to junctions and whether MAGI-1 modulates actin dynamics at junctions to further assess how MAGI-1 functions in a living organism.

Andrei Avanesov

Pete Chandrangsu

“Where to begin?: Promoter mutations that affect the transcription start site of rrnB P1”

Jason Peters

“Transcription Termination Factor Rho: Rise of the Nus”

Suraiya Haroon

“Investigating the Mechanism of the Memory of Acquired Stress Resistance”

Budding yeast pretreated with mild stress can survive what would otherwise be a lethal dose of stress. This phenomenon is termed acquired stress resistance (AqSR). Cells retain a memory of AqSR long after removal from the mild-stress treatment. For example, after an hour-long exposure to 0.7M NaCl (mild stress), cells washed and returned to stress-free conditions show resistance to doses over 2.0mM H2O2 (normally lethal dose) even after 4 generations. Protein synthesis is not required to maintain memory of AqSR once it has been acquired and this suggests long-lived proteins may be involved. A screen identified cytosolic catalase T, Ctt1p, as highly important for acquired H2O2 resistance, making it a likely candidate in the memory of H2O2 resistance. Our hypothesis is that long-lived Ctt1p contributes to maintaining the memory of H2O2 resistance.

Kate Baldwin

“The Role of the Conserved Protein, Cactin, in Arabidopsis Gravitropism and Development: An Exciting, Essential, and Unique Eukaryotic Gene”

CACTIN is a conserved gene found across eukaryotes and it is not a member of any gene family. Mutations in the CACTIN gene result in lethal phenotypes in Drosophila, C. elegans, and Arabidopsis. In flies, CACTIN plays a role in the NF-kB pathway, but there is no recognized NF-kB pathway in plants or C. elegans. Preliminary studies using Arabidopsis suggest that CACTIN plays a critical role in embryogenesis and possibly a part in root gravitropism. We are studying CACTIN in Arabidopsis, as well as examining its functional conservation between plants and animals. We hope to uncover the function of this important and mysterious gene.

Benjamin Schmidt

“Regulated splicing of C. elegans rsp mRNAs produces variants subject to nonsense mediated mRNA decay (NMD)”

Wei Shen

“Autophagy promotes synaptic growth in Drosophila”

Xu Chen

“Neuropeptide Signaling Regulates Larval NMJ Development in Drosophila”

Erica Andersen

“Axon Guidance in Zebrafish Sensory Neurons”

Marcus Miller

“Proteomic Analysis of the Heat Shock SUMOylome”

The covalent attachment of Small Ubiquitin-like MOdifier (SUMO) proteins to other intracellular proteins is an essential regulatory process in most eukaryotes. Despite this fact, little is known about the function of SUMO or the identity of most of its targets. In addition, one of the more striking phenomena associated with the SUMO pathway, its dramatic up-regulation in response to stress, remains unclear. Whereas most of the SUMO pool is in the free form under non-stressed conditions, various abiotic stresses (e.g., heat shock and chemical insults) induce its attachment to other cellular proteins in a rapid yet reversible reaction. To help catalog proteins affected by SUMOylation in Arabidopsis, we have developed an affinity method to enrich for the SUMO conjugates in plants, which can then be identified by tandem mass spectrometry. This method involves the complete replacement of the SUMO1 and SUMO2 isoforms with various tagged forms using an artificial SUMO1 transgene to rescue the normally lethal sum1-1 sum2-1 double mutant. The transgenic SUMO was also modified to contain an arginine near the C-terminal glycine. This novel trypsin cleavage site can then be detected by mass spectrometry as a footprint on the target peptide bearing a short C-terminal segment of SUMO attached via an isopeptide bond to the modified lysine. Using this affinity approach, we have developed an in-depth list of Arabidopsis proteins subjected to SUMOylation in vivo.

Jamie Elliott

“Early Onset Sleep Dysfunction in Drosophila Models of Neurodegenerative Disease”

Impaired sleep is a common phenotype among human patients suffering from neurodegenerative disease. The growing appreciation for the role of sleep in neuronal plasticity suggests that impaired sleep in these patients may contribute to the hallmark neuropsychological symptoms of neurodegeneration, including problems with learning, memory, and cognition. Drosophila is an ideal system in which to pursue the relationship between sleep and neurodegeneration, as Drosophila models of many neurodegenerative disease display phenotypes that are analogous to those found in human patients. Drosophila models of Alzheimer’s disease (AD), Huntington’s disease (HD), and Parkinson’s disease (PD) all display impaired sleep early in the course of disease progression. Neuronal expression of human A?42 results in reduced sleep and reduced nighttime sleep consolidation. RNA interference targeted at parkin, dj1?, or dpink1 RNA can be used to model PD, and also results in reduced sleep and reduced nighttime consolidation. In the AD model, sleep phenotypes can be reversed with pharmacological treatment with 3-iodo-tyrosine (3IY), a tyrosine hydroxylase inhibitor. The presence of sleep impairment in Drosophila neurodegenerative disease models, and the ability to reverse that impairment, makes it possible to test the role of sleep in the severity of neurodegenerative phenotypes. The observation that sleep impairment appears early in these Drosophila models also suggests that sleep impairment may play a causative role in neurodegeneration itself.

Namrata Asuri

“Characterization of Mechanisms and Molecules Involved in Zebrafish Rohon-Beard Peripheral Axon Guidance”

Anthony Studer

“The functional analysis of the teosinte branched gene”

Beth Dumont

“Little Mouse on the Prairie: Recombination Rate Variation in Natural and Laboratory Rodent Populations”

Talline Martins

“The genetic and molecular basis of discrete floral patterns in Clarkia gracilis”

Kirk Burkhart

“Forward and Reverse Genetic Screens Reveal Novel Nuclear RNAi Machinery”