• 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 LOOK FOR A PED HOMOLOG IN MAN?

2 1 Qa-2

α3

GPI LINKAGE

2 1

6 a.a. tail

HLA-G

α3

(NO GPI LINKAGE)

• 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?

Why use particle tracking/laser tweezers to track HLA-G?

Model of Phospholipid Bilayer making up cell membrane

Outer Leaflet of bilayer

Phospholipid Molecules

Hydrophobic Lipid Tail

Hydrophilic Head group

Cholesterol

FLUID MOSAIC MODEL OF CELL PLASMA MEMBRANE

SATURATED PHOSPHO LIPID(more ordered, less fluid)

UNSATURATED LIPID(more fluid in membrane)

GPI-LINKED PROTEIN

GPI-LINK IN OUTER LAYER OF BILAYER

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?

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

MAPPING THE SINGLE PARTICLE TRACK IN A CELL MEMBRANE

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.

What is an optical trap?

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.

Optical trap or ‘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

Moving particles around with the laser tweezers

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

• 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 1M AT 220C• AB COATED BEADS MEET OBSTACLES EVERY 3-4M @ 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