the role of sphingolipids in lipid raft function in paramecium tetraurelia
DESCRIPTION
The Role of Sphingolipids in Lipid Raft Function in Paramecium tetraurelia. Tyler Picariello 12/7/10. Outline. Background Model Organism Cilia and Lipid Rafts Methods Expected Results. Paramecium Background. - PowerPoint PPT PresentationTRANSCRIPT
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The Role of Sphingolipids in Lipid Raft Function in Paramecium tetraurelia
Tyler Picariello12/7/10
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Outline
• Background– Model Organism– Cilia and Lipid Rafts
• Methods
• Expected Results
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Paramecium Background
• Paramecium tetraurelia is a ciliated eukaryotic organism approximately 100-150m 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
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Cilia Background
http://www5.pbrc.hawaii.edu/allen/ch01/04-pm700521-14.html
0.2m
Image courtesy of Megan Valentine
100 nm
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Lipid Rafts and their Functions
Adapted from http://www.ncbi.nlm.nih.gov/books/NBK26892/
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Paramecium lipid composition and the Synthesis of Sphingolipids
• P. tetraurelia has a unique lipid composition, especially in the ciliary membrane
Lipids % Weight of Cell % Weight of Cilia
Cholesterol 3.6 5.2
Choline Sphingolipids 2.1 2.5
Ethanolamine Sphingolipids
3.8 15.5
Kaneshiro, 1987)
http://www.biol.unt.edu/~chapman/research%20projects/cotton/metabolic_pathways.htm
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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
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HypothesisDisruption 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 AimTo 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.
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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
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RNAi by feeding
L4440
HT115
Feed paramecium
Ds RNA
RNAi construct
SPT gene
Adapted from Haddon Pantel and Mellissa Donovan
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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
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Density Gradient Centrifugation
• Used to analyze the distribution of raft associated proteins in RNAi and control cells
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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
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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
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Questions?