MSc Exit Seminar- Aditi Aggarwal
Effect of thermal stress on the intracellular localization of constitutively expressed heat shock protein HSPA8 (Hsc70) in differentiated and undifferentiated cultured human neuronal cells
Heat shock protein HSPA8 (Hsc70) is a constitutively expressed member of the Hsp70 multigene family that is abundantly expressed in neuronal cells. Previous studies in our laboratory suggest that HSPA8 may play an important role in neuronal pre-protection against cellular stress. This thesis highlights the importance of HSPA8 and its role as a fast responder to cellular stress in differentiated human SH-SY5Y neuronal cells. The effect of thermal stress on the intracellular localization of HSPA8 is compared in differentiated and undifferentiated neuronal cells. HSPA8 rapidly translocated into the nuclei of differentiated neuronal cells after heat shock and co-localized at transcription sites with DNAJB1 (Hsp40) and HSPH1 (Hsp105α) components of the protein disaggregation/ refolding machine. The rapid targeting and assembly of an HSPA8 based disaggregation/ refolding machine acts as a nuclear protective mechanism in differentiated neurons without the time lag needed to induce stress-inducible Hsp70.
Supervisor: Prof. Ian Brown
MSc Exit Seminar- Stefan Schuetz
“Characterizing the basis of strigolactone perception by HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2”
Abstract:
The HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) α/β hydrolases likely play a critical role in the life cycle of parasitic plants of the genus Striga. Several of these hydrolases serve as receptors for strigolactones (SLs), a class of compounds exuded by the roots of some plants, including many economically significant crops. Upon perception of minute levels of SLs, Striga seeds germinate and parasitize the nearby host, often leading to massive losses in crop yields in affected regions. We have produced a series of mutant variants of the Arabidopsis thaliana homolog of HTL/KAI2, which is only weakly responsive to SL. By substituting certain key amino acids in the protein’s active site, we have created a receptor conferring increased Arabidopsis germination under inhibitory conditions in the presence of SL. Additional study of this mutant receptor may offer insight into the biochemical basis of Striga’s SL sensitivity.
Supervisor: Prof. Shelley Lumba
MSc Exit Seminar -Paige O’Leary
Induction of Heat Shock Proteins in Differentiated Human SH-SY5Y Neuronal Cells by Caffeine and Cafestol
Abstract:
During aging, protein quality control fails, leading to the accumulation of misfolded, aggregation-prone proteins which are characteristic of neurodegenerative diseases for which few effective therapies exist. Enhancement of protein quality control mechanisms is a potential treatment strategy, specifically upregulation of heat shock proteins (Hsps) that function to counteract misfolded proteins. Non-toxic inducers are required that can be ingested regularly and cross the blood-brain barrier to trigger Hsp induction in neurons that are affected in neurodegenerative diseases. Caffeine is a non-toxic compound taken daily that crosses the blood-brain barrier. The effects of two coffee components namely, caffeine and cafestol, are examined on the induction and intracellular localization of Hsps in differentiated human neuronal cells grown in tissue culture. Cafestol, but not caffeine, induced HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which localized to the nucleolus where these Hsps could protect ribosome biogenesis, a critical feature of neuronal protein synthesis.
MSc Exit Seminar- Alicia Harracksingh (Senatore lab)
Exploring Protein Interactions between CaV2 Calcium Channels and Pre-synaptic Scaffolding Proteins MINT and RIM in Invertebrates
During synaptic transmission, voltage-gated calcium type-2 (CaV2) channels mediate transient influxes of Ca2+ into the cytoplasm. This event activates synaptic vesicle (SV) calcium sensors, which facilitates docking, priming and fusion at the plasma membrane, and the subsequent release of SV contents into the synaptic cleft. A direct interaction has been proposed to tether CaV2 channels and SVs in nanometer proximity, primarily mediated by scaffolding proteins that bind to the distal C-terminus of CaV2 at a conserved PDZ-ligand domain. Here, we explored interactions between CaV2 and the scaffolding proteins RIM and MINT in vitro in the arthropod Drosophila melanogaster and the cnidarian Nematostella vectensis to understand the evolution of the synapse through conservation of CaV2 channel protein structure- function. We found that interactions between these proteins are conserved and occur at similar domains and ligand domains, which suggest that the CaV2-MINT and CaV2-RIM interactions serve as important evolutionary adaptations for the emergence of synapses.
MSc Exit Seminar- Yan Ling Iris Chiu (Chang Lab)
Functional characterization and molecular evolutionary analyses of dim-light adaptations in visual pigments and interactions with arrestin
Abstract:
The visual system of dim-light dwelling/nocturnal species are known be to highly adaptive due to the importance of vision for survival. Rhodopsin and cone opsins are responsible for dim-light and colour vision, respectively, and mediate photon absorption which results in isomerization of the retinal chromophore and activation of the visual pigment. This triggers the downstream visual transduction cascade which is subsequently inhibited by the binding of the signaling protein arrestin. Here, I investigated different aspects of dim-light adaptation using a combination of computational and experimental approaches. The first study investigated arrestin-rhodopsin complex formation relevant to a hypothesized photoprotection mechanism in dim-light vertebrates. Putatively adaptive mutations from dim-light animals identified in comparative analyses were found to enhance the ability of arrestin-rhodopsin complexes to sequester toxic retinal chromophore when experimentally assayed in vitro. The second study analyzed the selective pressures across the visual pigment gene RH2 of teleost fishes and found a positively selected site with unusual amino acid substitutions in the deep-sea northern lampfish. When assayed experimentally, substitutions at this site showed altered kinetics, with implications for better photosensitivity and cold adaptation. Overall, my findings provide insight into the less-well studied aspects of dim-light adaptation and show the significance of investigating the visual system as an interdisciplinary study involving evolution, biochemistry and molecular biology.
MSc Exit Seminar-Thomas Dodsworth
Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line
Teneurin C-terminal associated peptide (TCAP) is an ancient and conserved bioactive peptide that is evolutionarily related to corticotropin releasing factor (CRF). Recently, synthetic TCAP-1 was shown to increase cellular energy availability and alter contractile performance in rodent skeletal muscle. However, the exact receptor signalling mechanism through which this occurs is unknown. Based on evidence of their interaction in vitro, I hypothesized that TCAP-1 signals through latrophilins—a family of adhesion G-protein coupled receptors—to elicit its effects in muscle. To test this, I knocked-down and knocked-out latrophilins in the immortalized mouse myoblast C2C12 cell line by small interfering RNA (siRNA) and CRISPR/Cas9 gene editing methods, respectively, and examined the efficacy of TCAP-1 in knockdown and knockout cells. I determined that latrophilin-1 is necessary for TCAP-1-mediated increases in intracellular calcium, NADH turnover, and PGC-1a expression. This establishes, for the first time, that the TCAP-latrophilin ligand-receptor pair has a functional role in skeletal muscle.
MSc Exit Seminar- Paige O’Leary (Brown Lab)
Induction of Heat Shock Proteins in Differentiated Human SH-SY5Y Neuronal Cells by Caffeine and Cafestol
During aging, protein quality control fails, leading to the accumulation of misfolded, aggregation-prone proteins which are characteristic of neurodegenerative diseases for which few effective therapies exist. Enhancement of protein quality control mechanisms is a potential treatment strategy, specifically upregulation of heat shock proteins (Hsps) that function to counteract misfolded proteins. Non-toxic inducers are required that can be ingested regularly and cross the blood-brain barrier to trigger Hsp induction in neurons that are affected in neurodegenerative diseases. Caffeine is a non-toxic compound taken daily that crosses the blood-brain barrier. The effects of two coffee components namely, caffeine and cafestol, are examined on the induction and intracellular localization of Hsps in differentiated human neuronal cells grown in tissue culture. Cafestol, but not caffeine, induced HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which localized to the nucleolus where these Hsps could protect ribosome biogenesis, a critical feature of neuronal protein synthesis.
PhD Exit Seminar- Afif Aqrabawi
An Olfactory Memory Circuit
Episodic memory is defined simply as memory for what happened, when, and where. The hippocampus mediates episodic memory and represents contextual information using the parameters of space and time, including where an event unfolded and the sequential order of related events. Episodic recollections are characterized by rich multisensory details, yet the mechanisms underlying the reinstatement of these non-spatiotemporal aspects of experience are unknown. In this thesis, we identified direct, topographically organized hippocampal projections to a poorly understood ring-like structure known as the anterior olfactory nucleus (AON). We demonstrated that manipulation of the hippocampal-AON pathway can influence odour perception and odour-guided behaviours. Selective inhibition of hippocampal-AON projections impaired mice in their ability to recognize odours associated with the spatial and/or temporal aspects of their environment. We also revealed that AON activity is generated by coincident olfactory and contextual inputs arriving from the olfactory bulb and hippocampus, respectively. Thus, we hypothesized that the AON acts as the physical repository for populations of neurons representing previously encountered odours within the context in which they occurred. The precise pattern of activity produced by the AON therefore composes the olfactory memory trace, or “odour engram”. To this end, we used a tamoxifen-inducible Cre recombinase system to control the timing of gene expression in the AON. In combination with chemo- and optogenetic tools, we manipulated tagged AON neuronal populations in a carefully designed set of behavioural paradigms. We found that AON activity is necessary and sufficient for driving the behavioural expression of specific odour memories, thereby establishing the AON as the long-term storage site for contextually-based odour engrams. This thesis represents the first demonstration of the neural substrate of odour memory in vertebrates, satisfying all criteria used for defining an engram. The ease and suitability of using olfaction will undoubtedly position the hippocampal-AON pathway as an ideal circuit model for investigating fundamental mnemonic and cognitive principles. Indeed, this model can become particularly important in translational research that may yet lead to the development of therapeutic targets for disorders of memory, such as Alzheimer’s disease.
De novo genes and the long-term direction of their subsequent evolution
Joanna Masel from the University of Arizona, Department of Ecology & Evolutionary Biology
Most of the work in Joanna's group these days is connected in some way to evolvability. Joanna is most interested in models that explicitly capture mechanistic constraints, whether from biochemistry, genetics, cellular biology, physiology, or ecology, and work out their evolutionary consequences. Specific interests at the moment include the robustness and evolvability of biological systems, the origins of coding sequences from non-coding ancestors, and the tension between relative and absolute competitions in evolution, ecology, and economics.
Host: Alan Moses
Sequence-function relationships in intrinsically disordered regions through the lens of evolution
PhD Exit Seminar
Taraneh Zarin (Moses lab)
Intrinsically disordered regions (IDRs) are regions of proteins that do not autonomously fold into stable secondary or tertiary structures. Though they defy the classical view of proteins as rigidly structured macromolecules, IDRs are widespread in living organisms, and are involved in a diverse array of functions. The majority of IDRs appear to be rapidly evolving at the level of the primary amino acid sequence, which makes it difficult to quantify evolutionary conservation and associate these regions with biological function using standard sequence analysis. The aim of my thesis research has thus been to understand evolutionary constraint and sequence-function relationships in IDRs. Using a functionally characterized IDR in the yeast protein Ste50, I first found that highly diverged amino acid sequences can encode conserved phenotypes in IDRs, showing that sequence divergence does not necessarily imply functional divergence in these regions. Using a phylogenetic comparative framework, I found that the net charge of the Ste50 IDR, rather than the precise amino acids, is a functional molecular feature that is preserved over evolution. I next expanded my evolutionary analysis of IDRs to the yeast proteome, and found that most highly diverged IDRs contain many molecular features that are preserved over evolution. I summarized the evolution of these molecular features with an "evolutionary signature" for each IDR, and found that groups of IDRs with similar evolutionary signatures are enriched for specific biological functions. I also found that IDRs with similar evolutionary signatures can rescue function in vivo despite negligible sequence similarity. Finally, I used these evolutionary signatures to train a statistical model, and found that they can be used to classify IDRs for a diverse set of biological functions. I identified the molecular features contributing to these functional predictions, and attributed distinct functions to specific IDRs in proteins with multiple IDRs. Overall, this work shows that there is rich functional information in IDR sequences, and that this information can be revealed through evolutionary analysis.