Doctor of Philosophy in Cell and Developmental Biology (PhD)
Characterisation of novel targets of metabolic acid stress resistance in malignant cells
We currently have projects in the areas of genetic networks, cell signalling, membrane contact sites, cell polarity, cancer metabolism and autism. We use multiple model systems to study these topics including budding yeast for genetic network analysis, model human cell lines for cell signalling and microscopy, and knockout mouse genetic models for in vivo functional analysis.
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Membrane contact sites between the endoplasmic reticulum (ER) and other organelles are present in all eukaryotic cells. Their roles in calcium signaling and transport between the ER and the plasma membrane (PM) or the ER and mitochondria are quite well understood, but the molecular mechanisms underlying their roles in lipid synthesis and transport remains unknown. In order to identify the importance of organelle-ER contact sites, I used Saccharomyces cerevisiae - a model organism that has proven to be a particularly informative for studying lipid-related cellular processes. Previously, we found a role for an ER anchor protein, Scs2, being important for PM-ER contact sites. Further, SCS2 interacts genetically with ICE2, an ER gene with unknown function. In Chapter 2, I investigated a role for PM–ER contact sites in regulating phosphatidylcholine (PC) synthesis and I found that Δscs2Δice2 cells are choline auxotrophs and PM–ER contacts are required for PC synthesis. Osh2 and Osh3, the oxysterol-binding protein homologues in yeast, rescued the choline auxotrophy phenotype of Δscs2Δice2 cells but did not restore pmaER, indicating that they may function with Opi3 in PC synthesis. In search for regulators of pmaER, we identified the phosphatidic acid phosphohydrolase Pah1 that seems to be involved in establishing pmaER, independent of its enzymatic activity. Finally, we proposed that PE to PC synthesis by Opi3 happens “in trans” at PM-ER contacts. In Chapter 3, I aimed to discover novel genes involved in PE synthesis/traffic from ER to mitochondria. By doing a genome-wide screen for CHO2, we identified genetic interactions between CHO2 and Emc proteins indicating that Emc proteins are important for PE metabolism and we proposed that Emc facilitates PS transfer from the ER to mitochondria for PE synthesis. In Chapter 4, I investigated for roles of SCS2 in polarized growth. I found a physiologically important function of the ER diffusion barrier, which is to restrict diffusion of the spindle from mother to bud until M phase. Scs2 interacts directly with the spindle capture protein Num1 and it prevents Num1 from diffusing from the mother into the bud during S and G2 phases.
Polarization of cellular membranes into domains is an important mechanism tocompartmentalize cellular activities within the membrane and establish cell polarity.Recent studies have uncovered that the endoplasmic reticulum (ER) is polarized bydiffusion barriers, which in neurons controls glutamate signaling in dendritic spines, butthe molecular identity of these diffusion barriers is unknown. In Chapter 2 we show thata direct interaction between integral ER protein Scs2 and septin Shs1 creates the ERdiffusion barrier in yeast. We uncovered a new ER-associated polarisome subunit,Epo1, which is required for the tethering of ER to septins. The human homologue ofScs2, VAP-B, also interacts with Shs1 in yeast indicating that the tether may beconserved. As mutations in VAP-B cause amyotrophic lateral sclerosis, loss of ERpolarization in dendritic spines is a potential mechanism underlying motorneurondisease.Synthesis of phospholipids, sterols and sphingolipids is thought to occur atcontact sites between the ER and other organelles because many lipid synthesizingenzymes are enriched at contact sites. In only a few cases have the enzymes beenlocalized to contacts in vivo and in no instances have the contacts been demonstratedto be required for enzyme function. In Chapter 3 we show that plasma membrane (PM) -endoplasmic reticulum (ER) contact sites in yeast are required for phosphatidylcholinesynthesis and regulate the activity of a key enzyme, Opi3, whose activity requires a lipidbinding protein, Osh3. Thus, membrane contact sites provide a structural mechanism toregulate lipid synthesis.