The University of Pittsburgh is a leading research university, and the discovery of new scientific knowledge is a major focus of
the Department of Biological Sciences. Research within the Department is aimed at understanding a broad range of biological
processes, from the molecular and biochemical to the organismal and population levels. The Department fosters a highly
stimulating and interactive environment for the more than 25 individual research programs, and provides a foundation for exploring
novel biological phenomena that require interdisciplinary approaches.
Recent research in the Jeffrey Hildebrand Laboratory
The fundamental challenge to cell and developmental biologists is to unravel the basic mechanisms that control the ability of a single cell (the fertilized egg) to divide and differentiate into the trillions of cells found in the human body. As these cells are being generated, they must adopt the correct function, shape, and position such that they can be 'packaged' in a specific manner to generate the body plan that is eventually seen in the adult organism. Research in the Hildebrand lab investigates how cells control their shape during vertebrate development and how these changes in cell morphology facilitate the formation of embryonic and adult structures and tissues. This is an important area of research as defects in these process lead to a myriad of human disease and birth defects. Our recently published work has focused on the role of the Shroom family of proteins in regulating specific cellular characteristics during vertebrate embryogenesis. In mice, the Shroom family is comprised of Shroom2, Shroom3, and Shroom4. All of these proteins function by both conserved and unique mechanisms to control cellular morphology and cytoskeletal architecture. Specifically, these proteins are targeted to distinct locales in the cell via direct interactions with the F-actin-based cytoskeleton. Once in these subcellular compartments, Shroom proteins control the organization of the actin cytoskeleton and the activity of contractile actomyosin complexes to regulate cytoskeletal organization and cellular morphology. Our work has also determined that the activity of Shroom proteins appears to be conserved throughout metazoan evolution as we have identified what we predict are invertebrate orthologs of these proteins in Drosophila (fruit flies), Ciona (sea squirts), and sea urchins.
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Recent research in the Karen Arndt Laboratory
Transcription elongation is the productive stage of transcription, when the RNA transcript is synthesized, and a critical regulatory point in gene expression. Mutations that impair transcription elongation factors or viral proteins that commandeer the cellular transcription elongation apparatus give rise to multiple human diseases, including AIDS and cancer. To elucidate the mechanism of transcription elongation by RNA polymerase II, Kathryn Sheldon, a recent graduate student in the Arndt lab, isolated novel mutations in the yeast Paf1 complex, a highly conserved transcription elongation factor. By exploiting these mutations in a genetic screen for functionally related proteins, Kathryn identified a new role for the Paf1 complex in coordinating RNA synthesis with transcription termination and RNA 3' end formation. This finding reinforced the multifunctional nature of the Paf1 complex and revealed a role for this complex beyond transcription elongation.
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Recent research in the Beth Stronach Laboratory
Drosophila 'faces' have two prominent appendages called maxillary palps (asterisks). Disrupting SLPR function causes loss or malformation of the palps among other gross morphological defects.
Research in the Stronach laboratory has shown that a particular protein kinase is selectively used for many cell rearrangements that accompany embryonic development and metamorphosis during the fruit fly life cycle.
Development of an embryo involves precise orchestration of cell division, cell differentiation and cell migration. Failures in developmental programs can lead to birth defects or death. By studying the effects of mutations in a gene required during Drosophila embryogenesis, the Stronach lab has linked the function of a protein kinase, SLPR, to essential rearrangement of cells which will form the larval epidermis. SLPR is a highly conserved protein kinase related to human Mixed Lineage Kinases implicated in transmitting information to cells about extracellular signals in their environment. Though we don't yet know to what signal(s) SLPR responds, our studies suggest SLPR is activated at multiple times during the course of Drosophila development to coordinate the movement of epithelial cells during tissue closure events. For example, slpr mutants have malformed or missing facial features, called the maxillary palps (figure 1), derived from the same ancestral origin (the maxillary segment) as the human upper lip. Interestingly, human cleft lip and cleft palate are examples of improper tissue closure. With continuing genetic and cellular studies, we hope to gain more insight into the molecular mechanisms by which the family of Mixed Lineage Kinases regulates developmental tissue morphogenesis and homeostasis.
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Recent research in the Beth Roman Laboratory
Imaging blood vessel formation in live zebrafish embryos. The basilar artery and associated arterial vessels, which underlie the hindbrain and are enlarged in zebrafish alk1 mutants, sprout from surrounding veins. Dorsal view, anterior to the left. By comparing formation of this arterial vessel system in wild type and alk1 mutant embryos, we are beginning to understand the role of Alk1 within the endothelium.Click here to load the video (2.3 MB file)
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by formation of direct connections between arteries and veins. These aberrant connections may rupture or form shunts, resulting in clinical manifestations ranging from recurrent nosebleeds to ischemic or hemorrhagic stroke. It is well established that HHT is caused by mutations that disrupt signaling through the endothelial-specific TGF type I serine-threonine kinase receptor, Alk1. However, neither the role of Alk1 within the endothelium nor the etiology of HHT lesions is understood. Dr. Roman has identified a zebrafish alk1 homozygous mutant, violet beauregarde (vbg ), which exhibits embryonic vessel malformations similar to those seen in HHT patients. Zebrafish embryos are externally fertilized and transparent, and they develop very quickly, making them an ideal system to use for studies of vertebrate development. Using time lapse confocal microscopy of vessel development in the live embryo, as well as in vivo molecular analysis of genes putatively regulated by Alk1, recent studies in Dr. Roman's lab suggest that endothelial cells lacking Alk1 activity exhibit aberrant migratory behavior, in part due to failure to downregulate a critical receptor for an important chemotactic molecule. These new insights into the role of Alk1 within the endothelium are contributing to a better understanding of the etiology of HHT lesions.
The movie to the right was obtained by imaging a live, wild type zebrafish embryo expressing nuclear-localized enhanced green fluorescent protein (nEGFP) under the control of the endothelial-expressed fli1 promoter. Thus, each green dot represents an endothelial cell nucleus, which can be followed over developmental time. Using a laser scanning confocal microscope, images were captured every 12 minutes between 26 and 34 hours post-fertilization.
Recent research in the Joseph Martens Laboratory
The analysis of genome-wide transcription in many organisms, ranging from yeast to humans, has yielded a common, yet surprising feature: widespread transcription across non-protein-coding regions (often termed 'junk DNA'). Using yeast as a model eukaryotic system, the Martens' laboratory is investigating how this transcriptional activity and/or its non-protein-coding RNA products may influence gene expression and other cellular processes. In our most recent work published in Genes and Development (November, 2005), we describe a new mechanism for turning gene expression off that requires the transcription of non-protein-coding DNA. When serine levels are high, the yeast Cha4 protein activates transcription from non-protein-coding DNA (SRG1) adjacent to SER3, a gene that encodes an enzyme required for serine biosynthesis. The act of transcribing SRG1 across the SER3 promoter interferes the binding of transcription factors to the SER3 promoter thus turning SER3 gene expression off. This work elucidates a physiological role for transcription of non-protein-coding in controlling gene expression. Moreover, our results demonstrate a mechanism by which transcriptional activator proteins, such as Cha4, can function as transcriptional repressors by inducing transcription of non-protein-coding DNA.
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Recent research in the Deborah Chapman Laboratory
Research in the Chapman laboratory focuses on how the somites, tissue that will give rise to the ribs, vertebrae, skeleton muscle and dermis, are generated during development. We use the mouse as a model system because of the excellent genetic and molecular tools available and because the mouse is a mammal, it can be used to model human diseases and congenital birth defects.
Our recently published work in Genesis showed that Tbx6, a transcription factor, functions both upstream and downstream of the Notch signaling pathway during somite formation. Mutations in mouse Tbx6 eliminate somite formation in the posterior of embryos and lead to embryonic death. However, less severe mutants can lead to fusions of ribs and vertebrae (see figure to the right), a phenotype similar to humans and mice bearing mutations in the Notch signaling pathway. Altogether, our research uncovered both genetic and biochemical evidence for the link between Tbx6 and Notch signaling and may contribute to understanding of human birth defects.
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Recent research in the Graham Hatfull Laboratory
Research in the Hatfull lab focuses on the mycobacteria and their phages (viruses that infect them). The mycobacteria include human pathogens such as Mycobacterium tuberculosis , the causative agent of TB, and there is a need for better diagnostic, preventative and therapeutic approaches to control this disease. More than 30 complete mycobacteriophage genomes have been sequenced and employed as toolboxes for the development of a facile genetic system for the mycobacteria. In one specific recent advance, genes have been identified in one of these phages that promote elevated levels of homologous recombination in mycobacteria. Expression of these genes in mycobacteria stimulates allelic exchange with exogenously introduced linear DNA, enabling the rapid construction of gene replacement and gene knockout mutants. This work appears in the December 2006 online version of Nature Methods.
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Recent research in the Tia-Lynn Ashman Laboratory
Recent work on a global scale has shown that areas with the greatest plant biodiversity suffer the greatest pollen limitation and thus the lowering their chances of reproducing.
This work suggests that plants in these biodiversity 'hotspots' , which contribute substantially to the world's products and ecosystem functioning, may be the most at risk of extinction in the face of habitat destruction and global declines in pollinator diversity.
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Recent research in the Kirill Kiselyov Laboratory
Lysosomes are small vesicular organelles whose function is similar to that of the human digestive tract and involves digestion and absorption of food. Lysosomes also participate in autophagy, a process of digestion by a cell of its own effete organelles. Mutations in proteins that comprise lysosomal digestive machinery cause lysosomal storage diseases, the rare genetic disorders that render the cells unable to degrade lipids and/or proteins and cause them to accumulate undigested material. Lysosomal storage diseases lead to developmental delays, motor deficiencies and cognitive disabilities. There are indications of neuronal death in lysosomal storage diseases, although our understanding of the mechanisms of cell death in these diseases is incomplete. The Kiselyov’s lab has recently published a new model of cell death in lysosomal storage diseases that emphasizes housekeeping function of lysosomes. According to this model, lysosomal deficiencies in lysosomal storage diseases affect autophagy of effete mitochondria and result in accumulation of fragmented dysfunctional mitochondria. In addition to their role in energy production, mitochondria are actively involved in buffering cytoplasmic calcium, an excess of which is well known to cause apoptosis. A decrease in calcium buffering by mitochondria in cells affected by lysosomal storage diseases makes the cells vulnerable to pro-apoptotic effects of calcium, which may explain cell death in lysosomal storage diseases.
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Recent research in the Walter Carson Laboratory
Many non-native plant and animal species are accidentally or intentionally introduced into the US each year. Some of these exotic species can become invasive pests forming dense stands that threaten local biodiversity. Many scientists have argued that these exotic species are ecologically unique. Our research, however, has demonstrated the exotic and native species respond to broad geographic gradients in very similar ways, suggesting that exotic species are less unique than previously thought. Furthermore, our work shows that areas that were good for native species are even better for exotic species. The mechanism for this last result remains unexplained.
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Recent research in the Jeffrey Lawrence Laboratory
Rhodopseudomonas palustrus
Bacterial chromosomes are not merely collections of genes. On the contrary, there is structure embedded within and between genes that is critical for directing proper chromosome replication and segregatioin. Heather Hendrickson has discovered a class of sequences - termed Architecture IMparting Sequences, or AIMS - that increase in abundance on leading strands and decrease in abundance on lagging strands. AIMS are found in virtually all bacterial genomes. Some AIMS have been shown to be the binding site for the FtsK translocase, which pumps DNA into daughter cells during the process of cell division. This gradient of selection for AIMS shows that there are targets for natural selection above the level of the gene - that is, at the level of the chromosome arm, or replicore.
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Recent research in the William Saunders Laboratory
The Quintyne et al. manuscript identifies a novel requirement in the formation of multipolar spindles, a major divisional defect of cancer cells. We show that in addition to extra spindle poles, the malignant cell must inhibit an intrinsic clustering mechanism that holds the extra poles together. Spindle pole clustering is dependent on the microtubule motor dynein. Overexpression of a dynein-associated protein called NuMA in the cancer cells, inhibits clustering allowing extra poles to separate into a multipolar spindle. This abnormal structure divides the chromosomes irregularly, leading to genetic instability, a common feature of malignant cells.
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Recent research in the Michael Grabe Laboratory
Voltage-gated ion channels are essential to several fundamental biological activities such as the contraction of the heart and the generation of electrical stimuli throughout the nervous system. Given these critical roles, it is not surprising that mutations in these channels are linked to human diseases of the brain (epilepsy), ear (deafness), and heart (arrhythmia), and developing cures for these diseases requires a molecular understanding of the channels involved. A recent paper co-first authored by Assistant Prof. Michael Grabe has made steps toward elucidating how these channels open and close. Using a combination of high-throughput yeast screening and molecular modeling, they determined a down state structure of the voltage sensing domain of a voltage-gated potassium channel. This work reveals that the voltage sensing domains undergo large motions to open and close the channel and that the highly charged S4 segments of the channel are sandwiched between the center of the channel and outer helices both hotly debated topics in this field.
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Recent research in the Valerie Oke Laboratory
groEL1 (top) and groEL3 (bottom) in the root nodule
The groEL gene in bacteria encodes a chaperone that helps proteins fold properly. Although many bacteria, like E. coli , only have one copy of this gene, approximately 20% of the fully sequences bacterial genomes contain multiple copies. What is the function of the different versions? Are the genes differentially regulated to provide the chaperone at different times/levels or do the genes encode chaperones with different substrate specificities?
We are studying five groEL genes that are present in Sinorhizobium meliloti . This bacterial species interacts symbiotically with legume plants by living inside root nodules and fixing nitrogen for the host. Recent data indicates that groEL1 is the housekeeping gene because it is expressed at high levels, is sufficient for cell viability, and is necessary and sufficient for successful symbiosis. groEL5 , on the other hand, is specialized for stress response.
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Recent research in the Stephen Tonsor Laboratory
As environments change, the integration of key physiological traits is likely to change as well. In this study we explored the effect of increasing atmospheric CO2 on the integration of carbon, nitrogen and water use, demonstrating the shifts in their ultimate effects on reproductive fitness across a gradient of atmospheres.
We grew a large variety of wild-collected Arabidopsis thaliana ecotypes in five CO2 concentrations that spanned the range from typical interglacial Pleistocene concentrations to unprecedented levels expected in the next century. We showed that the effects of carbon uptake and use variation decline in their effects on fitness while the effects of N and H2 O increase in importance as atmospheric CO2 concentration increases. The stoichiometry of plant resource use relative to the stoichiometry of supply is thus likely to play a central role in both interspecific community dynamics and intraspecific adaptive evolution in plants.
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Recent research in the Paula Grabowski Laboratory
Alternative pre-mRNA splicing generates widespread transcript diversity by specifying the inclusion or exclusion of sequences encoding protein functional domains. We are interested in understanding how these mechanisms are coordinated in the nervous system to tailor the structure of protein molecules for their specific roles at the synapse relating to neuronal survival, communication, learning and memory.
Alternative splicing in response to neuronal excitation: The NMDA (N-methyl-D-aspartate) receptor (R1 subunit) plays roles at the synapse that are essential for learning and memory, and we have been using this pre-mRNA as a model system to study splicing regulation in the brain. The N1 and C1 cassette exons of this pre-mRNA encode protein domains that allow the receptor to respond to extra cellular interactions or participate in intracellular signaling. Using in vivo splicing assays and computational approaches, we have recently identified a sequence code that specifies the silencing (or, skipping) of a variety of exons throughout the human and mouse genomes. Exons containing this code are silenced by the binding of the splicing regulator hnRNP A1 to the RNA portions of this code. The C1 cassette exon responds to neuronal excitation by adjusting its splicing pattern toward increased exon skipping, and we have shown that this splicing response in primary neurons requires the presence of the identified sequence code. L-type Calcium Channels and the NMDA receptor itself are important for this response. Current efforts are aimed at understanding the mechanisms and biological rationale for these effects. How are events at the cell membrane communicated to the splicing machinery in the nucleus to cause the splicing changes observed during neuronal excitation? How widespread are these effects across the transcriptome? Why do these inducible splicing changes occur in neurons? An additional dimension of our work involves the identification of alternative splicing defects associated with neurological disease and cancer. We are working to understand, for selected examples, the underlying mechanisms responsible for abnormalities in regulation as a first step toward developing strategies to correct splicing defects in vivo .
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Recent research in the James M. Pipas Laboratory
Recent work in the Pipas lab (Oncogene, in press) has shown that the expression of SV40 TAg in intestinal epithelial cells results in a significant decrease in the levels of mRNAs encoding several drug metabolizing/detoxifying enzymes and transporters from the P450 pathway. The corresponding proteins are responsible for the metabolization, processing and final elimination of toxic compounds , including carcinogens and drugs. Components of this pathway are normally expressed in the liver and small intestine, and their levels increase after exposure to xenobiotics. We have found that TAg blocks both the endogenous levels of P450 components as well as the induction of these mRNAs by xenobiotics. Furthermore, this ability requires an intact LXCXE motif which binds to and inactivates the retinoblastoma (Rb) family of tumor suppressors in TAg suggesting that inactivation of this family plays a role in the process.
These results indicate that a functional Rb pathway in the intestine is necessary for the expression of the detoxification system used to clear carcinogens. The loss of this tumor suppressor or the alteration of its pathway, occurring in most human cancers, might change the susceptibility to chemical injury and tumor progression. We are actively investigating the connection between Rb, tumorigenesis and the P450 detoxification pathway and its possible significance in the metabolism and mechanism of action of both carcinogens and prescription drugs.
Recent research in the Rick Relyea Laboratory
Pesticides have become a ubiquitous component of the natural world to control the pests that impact human crops and human health. However, in assessing the risk that these chemicals pose to other (non-target) organisms, current regulations only require the testing of a small number of 'model organisms' including a fish, a zooplankton, a mammal, and a bird. For historical reasons, amphibians and reptiles are not tested. In the Relyea lab, we examine how pesticides affect amphibians via both direct toxic effects as well as more indirect effect that cascade through a food web. One of our most striking discoveries has been the discovery that Roundup®, the #1 herbicide in the U.S., is highly toxic to tadpoles at concentrations that can be found in nature. This work highlights the importance of testing pesticides on amphibians because we can discover highly lethal effects on a group of animals that are of current conservation concern.
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Recent research in the Anthony Schwacha Laboratory
Eukaryotic cells coordinate various essential functions to preserve the integrity of their genetic information. One site of co-regulation is the DNA replication fork - a mobile multi-component protein/DNA machine that not only is responsible for the regulation and achievement of genome duplication, but also coordinates sister chromatin cohesion and nucleosomal remodeling. The linchpin of this process is the replicative helicase, a molecular motor that both advances the protein components of the replication fork and unwinds duplex DNA ahead of the DNA polymerase. The presumptive eukaryotic replicative helicase is the minichromosome maintenance (MCM2-7) complex, a heterohexamer of six essential and highly conserved proteins. However, despite good in vivo evidence for their role in replication fork progression, the complex historically has lacked helicase activity in vitro . The Schwacha lab is pursuing both the mechanism and regulation of this fundamental cellular component by a variety of biochemical and genetic approaches. Recently, a graduate student in the lab, Matthew Bochman, has for the first time successfully reconstituted the helicase activity of the MCM2-7 complex in vitro . In contrast to other helicases, our work indicates that the different subunits of the MCM helicase contribute different ATP-dependent functions to the complex; some subunits appear to be essential for helicase activity, while other subunits appear to be required for loading the helicase onto DNA. These studies will allow us to expand our studies of MCM mechanisms to examine how other essential components of the replication fork affect the function(s) of the complex.
Recent research in the John Hempel Laboratory
Aldehyde dehydrogenase is most widely known for its role in metabolism of beverage alcohol, but should really be thought of in the plural, as a large highly diversified superfamily of enzymes with at least 18 distinct families. With collaborators at the Pittsburgh Supercomputing Center, details of the chemical mechanism are becoming known through quantum mechanical/molecular mechanical (QM/MM) simulations. Most recently, these calculations pointed to the chemically reasonable yet baffling formation of an adduct between the catalytic thiol and nicotinamide C-4. Since this would represent a dead-end complex we reasoned that these results must have been due to an incorrect starting data set, but within months of this finding, crystallographic evidence for precisely that adduct was independently found (Tsybovsky et al, Biochemistry 46: 2917 (2007); these authors also demonstrated the reversibility of the adduct), greatly supporting the validity of our computational approach. In a recent publication, we show that with NAD alone, formation of the adduct can only occur with NAD in the 'hydride transfer' orientation and not the 'hydrolysis' position some 4 Ångströms distant. Further, since the coenzyme must cycle through the hydride transfer orientation as a necessary component of the catalytic cycle, we also found that adduct formation in the presence of substrate is blocked by protonation of the oxyanion intermediate. All results continue to indicate that the proton for this event comes from a most surprising source - the mainchain amide nitrogen of the catalytic cysteine residue, an event which we believe is promoted by transient hydrogen bonding from a lysine residue in the second shell of residues surrounding the catalytic site to the carbonyl oxygen in peptide bond with the above amide nitrogen.
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Recent research in the Jeffrey Brodsky Laboratory
Approximately one-third of all proteins in eukaryotes enter the secretory pathway, and thus members of this diverse protein family interact with the endoplasmic reticulum (ER) during or soon after their synthesis. Because protein biogenesis is error-prone, mis-folded secreted proteins may be selected for ER associated degradation (ERAD). Not surprisingly, several human diseases arise from mis-regulation of the ERAD pathway. To better define how ERAD substrates are selected for degradation, the Brodsky laboratory has pioneered a number of in vitro and yeast expression systems. In a recent report in Cell, Nakatsukasa et al. have reconstituted the selection, ubiquitination, extraction, and processing of a model ERAD substrate. Through the use of this system, it was found that molecular chaperones recognize and then catalyze the interaction between ERAD substrates and an E3 ubiquitin ligase, and that a specific enzyme extends the ubiquitin chain to facilitate protein degradation. Interestingly, the ERAD substrate, which is an integral membrane protein, could be completely extracted from the lipid bilayer before it was processed by the proteasome. This event was catalyzed by an ATP-requiring, chaperone-like protein complex. Future efforts will dissect how ERAD substrates are targeted to the proteasome and how an integral membrane protein is maintained in solution en route to its destruction.
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