Current Research Projects
Dr. Michael Hendzel
E-Mail: michael.hendzel@ualberta.ca
398/498/499 Undergraduate Research Projects
The role of histone H1 in the DNA damage response
DNA double-strand breaks are the most dangerous form of DNA damage. Unless faithfully repaired, double-strand breaks can lead to the gain and loss of genomic material, apoptosis, oncogenic translocations, and genomic instability. Upon formation of a DNA double-strand break, a signaling cascade initiated by the phosphorylation of histone H2AX by the DNA damage activated kinase, ataxia telangiectasia mutated (ATM). A critical part of this signaling cascade is the generation of chains of ubiquitin covalently linked to one or more proteins found at the site of DNA damage. This forms a scaffold that is essential for the recruitment of proteins involved in the error-free repair mechanism, termed homologous recombination repair. The identity of the target protein has long been thought to be histone H2A. Recently, however, histone H1 was identified as the likely substrate for the formation of the K63-linked polyubiquitin scaffold. Our laboratory has a longstanding interest in histone H1. In our studies of another DNA damage-associated post translational modification, poly(ADP-ribosyl)ation, we found that histone H1 is rapidly displaced from DNA damage sites as a consequence of poly(ADP-ribosyl)ation. This result is seemingly inconsistent with a role of histone H1 as a scaffold for the assembly of proteins at sites of DNA double-strand breaks. We want to determine the dynamics of association of histone H1 with DNA double-strand breaks, how the association of histone H1 with sites of DNA damage is altered by the enzymes involved in the assembly of the ubiquitin scaffold, and how this relates to the binding properties of the proteins that bind to this scaffold, such as the breast cancer associated 1 (BRCA1) protein. This will be studied by introducing DNA damage into living cells expressing fluorescently tagged versions of H1 histones, mutant H1 histones, and fluorescently tagged proteins that are recruited to sites of DNA damage by association with the ubiquitin scaffold. Various kinetic techniques such as fluorescence lifetime imaging (FLIM) to detect protein-protein interactions, fluorescence recovery after photo bleaching (FRAP), and fluorescence correlation spectroscopy (FCS) will be used to quantify the dynamic properties of these proteins and their dependence on histone H1 and ubiquitin.
Dr. Paul LaPointe
E-Mail: paul.lapointe@ualberta.ca
398/498/499 Undergraduate Research Projects
Research in my laboratory is concerned with elucidating the molecular mechanism of action of the Hsp90 chaperone. Hsp90 is a highly conserved and essential protein in all eukaryotes. It is responsible for the folding and function of numerous signaling kinases, hormone receptors, transcription factors and other proteins. Hsp90 is also emerging as a key target in treatment of cancer, Cystic Fibrosis and other diseases. There are several undergraduate research projects available in my lab ranging from in vivo analysis of Hsp90 interactions with co-chaperones to biochemical/enzymatic analysis of Hsp90-mediated protein folding. Research projects can be tailored to suit the interests or goals of students. More information on my research can be found on my web page.
I can be contacted by email at paul.lapointe@ualberta.ca or by phone at 780-492-1804.
Dr. Thomas Simmen
E-Mail: thomas.simmen@ualberta.ca
398/498/499 Undergraduate Research Project
Endoplasmic Reticulum-Associated Rab GTPases and Neuronal Function
The organelles found in eukaryotic cells exhibit a unique protein composition that governs the functions of these organelles. Nevertheless, protein trafficking between organelles is imperative within cells. This is needed to mediate production and maturation of secretory proteins that require organelle-specific, localized modification of nascent proteins. In neurons, this aspect is particularly important, since neuronal cell bodies are extremely distant from their farthest extensions at the synapse. An important group of proteins that regulate secretion and intracellular trafficking are the Rab proteins: the largest subfamily of the Ras-like GTPases with at least 63 members in humans, but many more in several other organisms. Recently, our lab has identified about a dozen Rabs, which localize to the Endoplasmic Reticulum (ER). This is a surprising finding, since the ER is considered the point of origin of vesicular protein trafficking. We hypothesize that ER-associated Rabs perform functions, which are different from other Rabs and include ER-Golgi recycling, ER domain organization, ER structure and autophagosome formation. Independent research has identified some of these Rab proteins in the functioning of neurons, in particular in the maintenance of axons. The undergraduate project will aim to characterize one or more ER-associated Rab in terms of its function and, subsequently, in its significance for the maintenance of axons and neuronal survival.
Interested students should contact me by e-mail at thomas.simmen@ualberta.ca to discuss details.
Dr. Andrew Simmonds
E-Mail: andrew.simmonds@ualberta.ca
398/498/499 Undergraduate Research Project
Project Title: Characterizing a transcription factor complex that regulates heart muscle cell specification.
Approximately one percent of newborn infants manifest congenital heart malformation due to inherited mutations in one or more genes required for proper heart formation. We study two proteins (Sd/TEF-1 and Mef-2) that have critical roles in establishing a heart cell fate. There are multiple members of the human Sd/Tef-1 and Mef-2 protein families and it has been extremely difficult to identify co-factors that regulate their activity. However in the animal model system Drosophila melanogaster, there is only a single Sd/Tef-1 and Mef-2 protein family member required during heart formation. We have identified several potential novel co-factors and we are currently testing their role in heart formation and muscle differentiation. Due to the nature of this project, prospective students will need to have completed at least one 300-level course in Biology, Molecular Biology or Genetics. They will be using a combination of animal studies, visualizing of tissues in whole animals and transfection and culture of isolated cells to study muscle differentiation as it relates to the early events of heart formation.
Dr. Andrew Simmonds
E-Mail: andrew.simmonds@ualberta.ca
398/498/499 Undergraduate Research Project
Project Title: Post-transcriptional regulation of cell division by the Gw protein.
In eukaryotic cells, cytoplasmic processing (P-) bodies regulate directly regulate mRNAs. In mammalian cells, a core component of P-bodies is the Gw family of proteins. Using a combination of cultured cells as well as studies in whole animals, we are attempting to dissect the cellular roles and cytoplasmic events that regulate mRNAs. Specifically, we are using Drosophila to establish how Gw function is required for such critical cellular functions as regulating mitosis. With increased understanding of how this protein functions within the cell and how it, in turn, is regulated, our research will help with diagnosis and treatment of several poorly understood diseases that have been linked to the Gw homologues in humans. Due to the nature of this project, prospective students will need to have completed at least one 300-level course in Biology, Molecular Biology or Genetics. Additionally, preference will be given to academically superior third or fourth year undergraduate students looking to continue on to an MSc or PhD in Cell Biology. They will be using a combination of live-cell imaging, whole animal genetics, cell culture and molecular biology to study how basic cellular processes like mitosis and cell differentiation controlled by mRNA regulation.
Dr. Anastassia Voronova
E-mail: voronova@ualberta.ca
398/498/499 Undergraduate Research Projects
Project 1. Cell-cell communication between inhibitory neurons and neural stem cells for the generation of oligodendrocytes from neural stem cells.
Proper brain development and function requires neural stem cells (NSCs) to generate a specialized type of cell termed an oligodendrocyte at precise times, locations and in the right numbers. The purpose of oligodendrocytes is to produce myelin, an insulating material that performs vital functions in efficient neural information transmission and constitutes the brain white matter. The formation of oligodendrocytes and/or myelin is perturbed in neurodevelopmental disorders, such as autism spectrum disorder (ASD) and schizophrenia. White matter damage and inefficient remyelination occurs in neurological disorders like multiple sclerosis and white matter stroke. Therefore, it is important to understand how oligodendrocytes are generated from NSCs to not only understand the biology of neurodevelopmental disorders, but also to come up with novel pro-oligodendrogenic therapies for the injured brain. Our work identified a novel paracrine communication between NSCs and a specific type of neurons, inhibitory interneurons. Interneuron-secreted cytokine, fractalkine, promotes oligodendrocyte formation from NSCs in the developing mouse cortex. Several projects are available to study the role of interneuron-secreted molecules, such as fractalkine (FKN) and hepatoma-derived growth factor (HDGF), that drive the genesis of oligodendrocytes from NSCs. We will test how FKN and HDGF signalling in neural stem and/or oligodendrocyte precursor cells affects intracellular signalling pathways critical for oligodendrocyte differentiation from NSCs. This project will involve cultures of primary and/or immortalized precursor cells, shRNA-mediated knockdown analysis, polymerase chain reaction (PCR), immunofluorescent antibody staining and confocal microscopy.
Project 2. Epigenetic regulation of neural stem cell function and brain development.
Autism spectrum disorder (ASD) is highly prevalent in the Canadian population with an incidence of 1 in 66 children and is characterized by brain malformations and intellectual disability. Advancements in next generation sequencing have enabled identification of thousands of individual gene mutations in ASD patients, which cluster into three large groups: synaptic function, transcription and chromatin remodelling. While chromatin regulators constitute the majority of ASD risk genes, there is little understanding of how chromatin remodelling or epigenetic genes regulate the development of brain. Several projects are available to study the role of ASD risk epigenetic genes Ankrd11 (Ankyrin Repeat Domain 11), which affects histone acetylation and Kdm5b, which affects histone methylation. Histone post-translational modification is one of the hallmarks of epigenetic modification that has direct implications on global gene expression and cell behavior. We will test when perturbations of ASD risk epigenetic genes affects neural stem cell function and what is the chromatin-mediated mechanism of neural stem cell regulation by these genes. This project will involve cloning, isolation and cultures of primary mouse embryonic cortical precursor cells, immunofluorescent antibody staining and confocal microscopy. This project has the potential to evolve into a graduate project.
Dr. Richard Wozniak
E-mail: rick.wozniak@ualberta.ca
398/498/499 Undergraduate Research Projects
Project Title: Function of Flaviviridae viral RNAs in the host cell nucleus
Abstract: Zika, Hepatitis C, Dengue, and West Nile viruses of the Flaviviridae family infect hundreds of millions of people, causing widespread morbidity and mortality. A prominent example is the recently discovered congenital Zika syndrome characterized by severe microcephaly. Except for HCV, there are no approved anti-viral treatments for these viruses, despite decades of research. A central tenant of Flaviviridae biology, and one that defines therapeutic strategies, is that the virus life-cycle occurs within the cytoplasm of host cells. We have now directly challenged this dogma by showing, using highly sensitive and specific detection techniques, that the RNA genomes (vRNAs) of Zika and HCV enter and leave the host cell nucleus (see figure), and travel through the nucleus is required for virus production.
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Localization of +vRNAs at the indicated times in Zika infected cells detected by single molecule fluorescent in situ hybridization. +vRNAs determined to be outside (red) or inside (yellow) the nucleus (blue) are indicated by the colored spot. Bar=5 µm. |
This breakthrough requires a paradigm shift away from a cytoplasm centric view of Flaviviridae biology and a re-evaluation of how we study and combat these viruses. However, before we can leverage this knowledge for societal benefit (e.g. therapeutics), we must understand, at a mechanistic level, why these viruses engage nuclear processes, and how this benefits the virus. Our research will tackle these questions using highly innovative approaches that will allow us to construct dynamic and cell specific systems-level interaction networks between vRNAs and host factors. We expect these high-risk, high-reward endeavors will produce new knowledge and foster novel pan-Flaviviridae therapeutic opportunities.
Proposed student project: The undergraduate student will work with a graduate student and research associate in the lab. Their experiments will first focus on constructing Zika viruses where an RNA tag is inserted into the viral genome. The tagged Zika virus will then be produced in tissue culture cells. The tag in the virus genome, in conjunction with nuclear or cytoplasm proteins that bind the tag, will be used to either increase or decrease the nuclear amounts of the genome in infected cells. The consequences of shifting the cellular distribution of the viral genome on production of the virus will be examined. This work will involve teaching the student basic molecular cloning, cell transfections, fluorescence microscopy, and basic handling and analysis of the Zika viruses. Note, other potential projects are available and can be discussed.
Abstract: The nuclear envelope (NE) provides an environment for the proper regulation and organization of interacting chromatin. In budding yeast, NE-associated chromatin includes telomeres and silenced subtelomeric regions. Multiple mechanisms are employed to tether these chromatin regions to the NE and silence resident genes. Among the factors required for telomere association with the NE is Siz2, a SUMO E3 ligase (Ferreira, et al., 2011. Nature Cell Biology, 13(7), 867-74). We have investigated the role of Siz2 and sumoylation in telomere association with the NE. We found that Siz2 is predominantly distributed throughout the nucleoplasm during interphase, but is recruited to the NE during the early stages of mitosis, when newly replicated telomeres re-associate with the NE. Enrichment of Siz2 at the NE is dependent on its mitotic phosphorylation, a SUMO interacting motif within Siz2, and a nuclear pore complex protein, Nup170, which is known to interact with subtelomeric chromatin (Van De Vosse et al., 2013. Cell, 152(5), 969-983). In all cases, mutant cells which fail to recruit Siz2 to the NE results in telomere tethering defects. We hypothesize that NE recruitment of Siz2, during mitosis, functions to target specific proteins for sumoylation that are required for proper chromatin organization at the NE. Consistent with this model, we show that NE recruitment of Siz2 correlates with the mitotic specific sumoylation of several proteins. This includes Scs2, an integral membrane protein and member of the VAP protein family. Scs2 resides in the endoplasmic reticulum (ER) and the NE where it appears to function in phospholipid metabolism, endoplasmic reticulum/plasma membrane interactions, and telomeric silencing. We have shown that Scs2 is required for telomere association with the NE and our data suggests that Scs2 functions as a receptor for Siz2 on the inner nuclear membrane. Together these data suggests that Scs2 plays an essential role in the spatial and temporal control of sumoylation at the inner nuclear membrane, an important post-translational modification involved in chromatin organization.
Proposed student project: The undergraduate student will work with a graduate student and research associate in the lab. Their experiments will focus on the links between sumoylation, lipid metabolism, and the regulation of nuclear membrane structure. The student will use fluorescence and electron microscopy to examine changes in nuclear membrane structure in yeast mutants that alter the sumoylation of nuclear envelope proteins. Mutations in sumoylation that lead to nuclear membrane proliferation will be tested to examine how they affect lipid metabolism. This work will involve teaching the student basic molecular cloning, yeast molecular genetics, fluorescence microscopy, and basic protein analysis. Note, other potential projects in this area are available and can be discussed.