The Role of Sphingolipids in Lipid Raft Function in Paramecium tetraurelia

1. The Role of Sphingolipids in Lipid Raft Function in Paramecium tetraurelia Tyler Picariello 12/7/10

2. Outline • Background • Model Organism • Cilia and Lipid Rafts • Methods • Expected Results

3. Paramecium Background • Paramecium tetraurelia is a ciliated eukaryotic organism approximately 100-150m in length • Excellent model for studying ciliary lipids and proteins • Changes in membrane potential can be observed through changes in swimming behavior http://bioinformatica.upf.edu/2008/projectes08/Dy/paramecium_intro.jpg Pantel, Haddon; Undergraduate Honors Thesis, 2007

4. Cilia Background 100 nm Image courtesy of Megan Valentine 0.2m http://www5.pbrc.hawaii.edu/allen/ch01/04-pm700521-14.html

5. Lipid Rafts and their Functions Adapted from http://www.ncbi.nlm.nih.gov/books/NBK26892/

6. Paramecium lipid composition and the Synthesis of Sphingolipids • P. tetraurelia has a unique lipid composition, especially in the ciliary membrane Kaneshiro, 1987) http://www.biol.unt.edu/~chapman/research%20projects/cotton/metabolic_pathways.htm

7. Lipid Rafts in Paramecium • Lipid rafts in P. tetraurelia share important general raft properties • Resistant to cold non ionic detergent extraction • They are enriched with cholesterol, glycosphingolipids and GPI- anchored proteins • Paramecium lipid rafts can be further divided into Methyl--cyclodextrin sensitive and insensitive rafts

8. Hypothesis Disruption of sphingolipids, a key component of ciliary lipid rafts, through the depletion of the serine palmitoyltransferase (SPT) gene message will result in disruption of ciliary lipid raft formation. This will in turn disrupt Folate chemoattraction and ciliary calcium channel function. Specific Aim To study the effect of serine palmitoyltransferase mRNA depletion on lipid raft formation in Paramecium. SPT mRNA depletion will be achieved through the RNAi feeding method. I. The effects of SPT mRNA depletion on lipid raft organization will be analyzed by sucrose density gradient centrifugation. II. Study the effects of SPT mRNA depletion on Folate chemoattraction using T-Maze assays III. Study the effects of SPT mRNA depletion on ciliary calcium channel function using backward swimming assays.

9. RNAi Background • RNAi is a method used to down-regulate specific mRNA sequences • Double stranded RNA (dsRNA) introduced into the cell is cleaved into segments of 20-25 nucleotides in length (siRNA) by the enzyme Dicer • The guide strand of the siRNA is incorporated into the RISC complex allowing it to target and pair with the complementary mRNA sequence • This results in cleavage of the mRNA sequence and down-regulation of the specific gene product http://www.abcam.com/cms/displayImage.cfm?intImageID=21696

10. RNAi by feeding RNAi construct L4440 SPT gene HT115 Feed paramecium Ds RNA Adapted from Haddon Pantel and Mellissa Donovan

11. T-Maze Assay • Used to test attraction behavior • Control Solution: NaCl • Test Solution: Na2-Folate • Paramecium are allowed to swim for 30 minutes • Count the cells in each arm • Iche= # cells in test arm • total # of cells Control Arm Test Arm

12. Density Gradient Centrifugation • Used to analyze the distribution of raft associated proteins in RNAi and control cells

13. Backward Swimming Assays • Membrane potentials will be stabilized via exposure to KCl buffer • Cells tested in high potassium and barium chloride solutions as well as sodium chloride • Time spent swimming in reverse will be measured and is directly proportional to the number of functional Ca2+ channels present in the ciliary membrane

14. Expected Results • RNAi will result in the disruption of ciliary lipid rafts domains reflected in a shift in protein distribution in the sucrose gradient • Disruption of GPI anchored Folate binding proteins will result in decreased attraction to Folate in T-Maze Assays • Expect decreased backward swimming time due to defective voltage gated Ca2+ conductance

15. Questions?