Sally McFall et al. (2005) International Worm Meeting "The Development of Regulatable Transgene Expression Systems for Caenorhabditis elegans"

Rick Morimoto, Sally McFall, Cindy Voisine

[International Worm Meeting, 2005]

Caenorhabditis elegans is a powerful genetic model system used to study a wide range of biological phenomena due to its short life span, cell lineage map, fully sequenced and annotated genome, and relative ease of genetic manipulation. While C. elegans has a wealth of genetic tools, the availability of conditional promoters for the expression of exogenous genes is limited. To date, the system in wide use is the heat shock promoter, which has the consequence of up-regulating molecular chaperones and other stress responsive genes. Activation of a stress response during development or aging is certain to have consequences and potentially interfere with the ability to interpret experimental results. To address this limitation in the field, we are creating alternative regulatory expression systems for C. elegans. Initially, we have chosen to focus our efforts on the development of the Lac repressor system. The Lac repressor system was selected because it is highly utilized in other species, the application of components of this system have already been demonstrated in worms and this system can also be applied in a tissue specific manner. Once we have created a specifically controlled expression system, we will make the reagents available to the C. elegans community.

Cindy Voisine et al. (2007) International Worm Meeting "Investigating the Effects of HSP70 Overexpression in Maintaining Protein Homeostasis."

Cindy Voisine, Michael Schieber, Richard Morimoto, Sally McFall

[International Worm Meeting, 2007]

A hallmark of numerous age-related neurodegenerative diseases is the deposition of protein aggregates and inclusions in affected tissues. Despite the functional and structural diversity of proteins involved in these diseases, the shared characteristic of accumulation of misfolded proteins has led to a unifying hypothesis that the chronic expression of aggregation-prone proteins disrupts protein homeostasis. Cellular protein homeostasis is a balance between folding of proteins by molecular chaperones and removal of damaged proteins by the proteasome. Molecular chaperones, key components of the cellular folding machinery, interact with misfolded proteins and prevent aggregation. To identify additional pathways involved in preventing aggregation, our lab performed an RNAi screen identifying a functionally diverse set of 186 genes whose decrease in expression produced an earlier onset of aggregation in our C. elegans polyglutamine (polyQ) model. The mechanism by which these pathways disrupt protein homeostasis leading to polyQ aggregation is unclear. We hypothesize that specific chaperone activity is required for proper function of these cellular pathways. The chronic expression of aggregation-prone proteins may titrate a critical chaperone function. We are manipulating the levels of specific molecular chaperones in combination with RNAi against components of these cellular pathways and monitoring polyQ aggregation. Through overexpression of HSP-1, a member of the highly conserved HSP70 family, we will genetically identify the proteins and processes requiring HSP-1 activity to maintain cellular protein homeostasis.

Li-Jung Tai et al. (1999) International C. elegans Meeting "CeHSF and HSB-1: The heat shock response regulators"

Catherine Airey, Sue Fox, Tim Roytman, Sally McFall, Sanjeev Satyal, Richard Morimoto, Li-Jung Tai

[International C. elegans Meeting, 1999]

One of the key components of the stress response is the heat shock transcription factor whose activation leads to synthesis of heat shock proteins. We have analyzed the C. elegans HSF with the help of C. elegans genome project, and found the following features. First, the C. elegans genome appears to encode a single heat shock factor (CeHSF Y53C10A.12), unlike higher metazoans whose genomes encode multiple HSFs. Second, CeHSF encodes a 75 KD protein with a DNA binding domain (residues 87 to 196) and a trimerization domain composed of hydrophobic repeats (HR) A and B (residues 193 to 285). Although the overall identities of CeHSF to S. Cerevisiae (ScHSF) and human HSF (HsHSF) are 17 and 24% respectively, the identities of the DNA binding and trimerization domains to ScHSF and HsHSF are 23 and 40% respectively. Third, sequence analysis of CeHSF showed an apparent lack of HR-C, a regulatory motif of heptad repeats at the C terminus conserved in all known animal HSFs. HR-C has been shown to be important for keeping HSF in a latent monomeric state during unstressed condition. The fact that C. elegans heat shock response has been shown to be inducible, yet CeHSF lacks the HR-C to keep it inactive in normal condition indicates that the mechanism of CeHSF activation may be distinct from other known animal HSFs. Lastly, the EST of C. elegans HSF (EST yk485h11.5) binds specifically to the consensus heat shock element when expressed as a recombinant fusion protein in bacteria. Currently, we are making antibodies and deletion worms to study the expression and function of CeHSF. In the process of dissecting the regulatory mechanism of HSF, a heat shock binding protein (HSB-1) was cloned and shown to negatively regulate the heat shock response in both C. elegans and mammalian cell lines. The HSB-1 deletion worms have a 30% decrease in life span comparing to N2 wildtype worms. The role of HSB-1 in regulating the heat shock response and its interaction with HSF are being examined using overexpression and deletion worms, as well as mammalian tissue culture models. Satyal SH et. al. Genes and Dev. 1998, 12(13):1962-74. Stringham, EG et. al. Molecular Biology of the Cell. 1992, 3:221-233. Wu, C. Annu. Rev. Cell Dev. Biol. 1995, 11: 441-69.