Download Hücre Membran Yapısı - mustafaaltinisik.org.uk

WHY LOOK FOR A PED HOMOLOG IN MAN?
• PED GENE PRODUCT IS MHC CLASS 1B MOLECULE Qa-2
• Qa-2 CONTROLS RATE OF EARLY CLEAVAGE DIVISION
• Qa-2 INFLUENCES SUBSEQUENT EMBRYO SURVIVAL
• HUMAN EMBRYOS HAVE VARIED CLEVAGE RATES
• FASTEST CLEVAGE RATES GIVE BEST PREGNANCY OUTCOME
• PED PHENOTYPE APPEARS TO EXIST IN MAN
• WE BELIEVE HLA-G IS THE HUMAN Qa-2 FUNCTIONAL HOMOLOG
Why use particle tracking/laser tweezers to track HLA-G?
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HLA-G is its putative human functional homolog of murine Qa-2 protein
There are good sequence and structural homologies between the molecules
Neither has a cytoplasmic tail - How do they send signals into the cell?
Qa-2 has a GPI-Linkage to the cell membrane and is found in caveolae
HLA-G has no GPI linkage-localization in membrane not reported
Can both molecules associate with other components in the membrane
and still share a conserved signaling pathway?
• Can SPT/laser tweezers allow us to determine location HLA-G in the
cell membrane by evaluating the behavior of the molecule tagged with gold beads?
2
1
HLA-G
α3
2
1
Qa-2
α3
(NO GPI LINKAGE)
6 a.a. tail
GPI LINKAGE
Model of Phospholipid Bilayer making up cell membrane
Phospholipid
Molecules
Outer Leaflet of bilayer
Hydrophobic Lipid Tail
Hydrophilic Head group
Cholesterol
FLUID MOSAIC MODEL OF CELL PLASMA MEMBRANE
GPI-LINKED PROTEIN
GPI-LINK IN
OUTER LAYER OF
BILAYER
SATURATED PHOSPHO LIPID
(more ordered, less fluid)
UNSATURATED LIPID
(more fluid in membrane)
WHAT IS SINGLE PARTICLE TRACKING?
• SPT EXPERIMENTS FOLLOW BROWNIAN MOTION OF MEMBRANE PROTEINS
IN TERMS OF CHANGE IN POSITION OF AB-COATED BEADS BOUND TO THE
MOLECULE OF INTEREST
• CHANGE IN POSITION OF CENTROID OF A BEAD CAN BE TRACKED WITH
NANOMETER PRECISION
• PARTICLE TRACKS DERIVE FROM SEQUENTIAL VIDEO FRAMES OF A LABELED
CELL
• TRACKS GIVE INFORMATION ABOUT LATERAL DIFFUSION OF BEAD-LABELED
MOLECULES.
• SPT CAN REVEAL LOCATION OF MOLECULE IN PLASMA MEMBRANE e.g
IS MOLECULE LOCALIZED IN LIPID RAFT?
MAPPING THE SINGLE PARTICLE TRACK IN A CELL MEMBRANE
Lateral transport modes on the cell surface. (A) Transient confinement by
obstacle clusters, (B) or by the cytoskeleton, (C) directed motion, and (D) free
random diffusion (Jacobson et al Science, (1995) 268: p 1441
What is an optical trap?
You may not know what an optical trap is, but you've probably all seen something
like it. It's essentially a tractor beam like one from an episode of Star Trek, only on
a microscopic scale.
Optical trap or ‘laser tweezers’
In the geometric optics regime light
can be represented by light rays. Two
rays 1 and 2 of a laser beam focused
in f are shown. Upon hitting a
dielectric particle with refractive
index (np) greater than the refractive
index (nm) of the surrounding
medium the light rays are refracted.
The changes in light momentum for
the two rays result in forces F1 and F2
on the particle. The sum of forces F in
a focused laser beam drives the
particle back into the focus.
Moving particles around with the laser tweezers
• A single beam of tightly focused laser light creates an extremely high
electric field gradient in the vicinity of the focus.
• Similar to the force which draws a dielectric into the high field region of a
capacitor, a dielectric particle falling within the laser beam will experience a
force which is directed towards the focus of the beam.
•Provided the numerical aperture of the focusing optics is high, the so called
"optical tweezers" give rise to a 3-dimensional trap.
•The force due to the field gradient is sufficiently high to overcome the forces
due to both gravity and radiation pressure. The particles can be moved around
in three dimensions.
• If the particle in a membrane moves into a more ordered/less fluid part of
the membrane, it will ‘escape’ the optical trap
• Behavior of the particle can be used to evaluate the ‘topology’ of the local
membrane structure
WHAT’S THE BIG DEAL ABOUT CAVEOLAE
(A SUBSET OF LIPID RAFTS)?
• DEFINED BY:
• RESISTANCE TO SOLUBILZATION BY TRITON-X AT 40C
• LIGHT BOUYANT DENSITY
• ENRICHED IN GLYCOSPHINGOLIPIDS, CHOLESTEROL AND
LIPID ENRICHED MEMBRANE PROTEINS (E.G GPI-LINKED)
• CALVEOLIN-1 IS MARKER PROTEIN
• Qa-2 IS FOUND IN CAVEOLAE
• COMPARTMENTALIZATION OF SIGNALING ACTIVITIES
• RICH IN RECEPTORS AND INTRACELLULAR SIGNAL-TRANSDUCERS
• REMEMBER THAT SHORT TAIL ON MEMBRANE-BOUND HLA-G? EVEN WITH
GPI LINKAGE MISSING, DOES IT AGGREGATE IN CAVEOLAE LIKE QA-2?
• IF HLA-G AND Qa-2 BOTH AGGREGATE IN CAVEOLAE, WE CAN POSTULATE
THE EXISTANCE OF A COMMON/CONSERVED DOWNSTREAM SIGNAL
TRANSDUCTION PATHWAY, WHICH WILL THEN HAVE TO BE PROVEN
EXPERIMENTALLY
GPI-linked proteins aggregate in lipid ‘rafts’ in the cell
membrane when they are cross-linked on the surface
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Lipid rafts may or may not contain caveolae
Lipid rafts tend to have more ordered lipids, less fluidity than normal membrane
When a particle-tagged molecule moves into a raft, it ‘escapes’ from the laser trap
Particle diffusion co-efficient, barrier free path length, and resistance measurements
can be made
EXPERIMENTAL CONSIDERATIONS FOR SPT EXPERIMENTS
•TEMPERATURE
• AB COATED BEADS MEET OBSTACLES EVERY 1mM AT 220C
• AB COATED BEADS MEET OBSTACLES EVERY 3-4mM @ 340C
• TO FIX OR NOT TO FIX:
• CHEMICAL CROSS LINKING OF MEMBRANE PROTEIN TO CYTOSKELETON
COULD PREVENT LATERAL DIFFUSION THAT WOULD OTHERWISE OCCUR
• Fab OR IgG:
• IgG SEEMS TO GIVE REDUCTION IN MOBILITY IN SOME SPT EXPTS
• IgG MAY ALTER MOLECULE DISTRIBUTION IN MEMBRANE
• HOW TO BIND PROTEIN TO BEAD
• SOMETIMES CAN’T BE ATTACHED DIRECTLY TO BEAD
• APPROPRIATE PROTEIN CONCENTRATION (DENATURES ON BEAD)
• HIGH CONCENTRATION CAN CROSSLINK MOLECULES
• CROSS LINKING CAN CHANGE DISTRIBUTION
• SIZE OF BEAD INFLUENCES BEHAVIOR OF MOLECULES BEING TRACKED
• MANY EXPERIMENTAL CONTROLS MUST BE DONE TO VALIDATE RESULTS