Transcript Tyler`s Presentation

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
100-150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
Cilia Background
100 nm
Image courtesy of Megan Valentine
0.2m
http://www5.pbrc.hawaii.edu/allen/ch01/04-pm700521-14.html
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/me
tabolic_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
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 TMaze assays
III. Study the effects of SPT mRNA depletion on ciliary calcium channel function
using backward swimming assays.
RNAi Background
•
•
•
•
http://www.abcam.com/cms/displayImage.cfm?intImageID=21696
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 downregulation of the specific gene
product
RNAi by feeding
RNAi construct
L4440
SPT gene
HT115
Feed
paramecium
Ds RNA
Adapted from Haddon Pantel and Mellissa Donovan
T-Maze Assay
•
•
•
•
Control Arm
Test Arm
•
•
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
Density Gradient Centrifugation
• Used to analyze the distribution of raft associated proteins in RNAi and control cells
Cilia pellet mixed with 80% sucrose and TNE buffer to achieve a final concentration of 40%
Pellet overlayed with a 5%-40% continuous linear sucrose gradient
Tubes spun at 160,000 x g for 4 hours
Tubes divided into fractions and used for density analysis
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?