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

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

3. Paramecium Background
Paramecium tetraurelia is a ciliated eukaryotic organism approximately m in length
Excellent model for studying ciliary lipids and proteins
Changes in membrane potential can be observed through changes in swimming behavior
Pantel, Haddon; Undergraduate Honors Thesis, 2007

4. Cilia Background
100 nm
Cilium is derived from the basal body and projects outward from the cell
Motile cilia have the 9+2 arrangement of microtubule doublets forming the ciliary axoneme
Hyperpolarization of the membrane generates the power stroke propelling the cell forward, depolarization causes a reversal of the power stroke resulting in backwards swimming
Image courtesy of Megan Valentine
0.2m

5. Lipid Rafts and their Functions
Contain high levels of saturated fatty acids, sterols and sphingolipids. Saturated fatty acids tend to make these regions more tightly packed than the surrounding membrane. Raft domains interact with cytoskeletal elements, but can float freely in the lipid membrane
Multiple types of raft associated proteins. Some partition in these regions through protein lipid interactions (hedgehog-cholesterol bound). Others associate as a result of post translational modifications (folate receptor-GPI anchor bound)
May also be involved in helping to regulate Calcium homeostasis and membrane potential
Lipid rafts are normally disrupted by chelation of cholesterol using methyl-B-cyclodextran
Adapted from

6. 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)

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 Specific Aim
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.
Previous work has shown that treatment of Paramecium ciliary lipid rafts with Methyl-b-Cyclodextrin, to remove sterols, does not result in the disruption of ciliary lipid raft domains (Pan, 2008).

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 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

10. RNAi by feeding RNAi construct L4440 HT115 Feed paramecium Ds RNA
SPT gene
HT115
L4440 has two T7 promoters flanking the construct region making it good for producing large amounts of dsRNA
HT115 bacteria are RNAse III deficient so they cannot degrade the RNA that is produced
Do not know how the dsRNA is incorporated into Paramecia, just that we see downregulation
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
TNE buffer = Tris base, NaCl and EDTA
Acts as a lysis buffer

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
When lipid rafts are associated with cytoskeleton they float in high buoyant density region disruption will cause them to float in low buoyant density. Non associated rafts have low buoyant density and disruption will cause a shift in the protein distribution in the specific fraction.
High potassium: short backwards swimming due to reduced voltage activated calcium conductance (Channels exclusively in the cilia). Since less channels are present in the membrane, less calcium would be entering the cell leading to shorter backwards swimming in high potassium
High sodium: sodium depolarizes the cell but no v gated Ca2+ channels to aid in depol so shorter backwards swimming

15. Questions?