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

• 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

Cilia Background

http://www5.pbrc.hawaii.edu/allen/ch01/04-pm700521-14.html

0.2m

Image courtesy of Megan Valentine

100 nm

Lipid Rafts and their Functions

Adapted from http://www.ncbi.nlm.nih.gov/books/NBK26892/

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

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

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.

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

RNAi by feeding

L4440

HT115

Feed paramecium

Ds RNA

RNAi construct

SPT gene

Adapted from Haddon Pantel and Mellissa Donovan

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

Density Gradient Centrifugation

• Used to analyze the distribution of raft associated proteins in RNAi and control cells

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

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

Questions?