H

1
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

2
Why use particle tracking/laser tweezers to track
HLA-G?

• 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?

3
Model of Phospholipid Bilayer making up cell
membrane
Phospholipid Molecules
Outer Leaflet of bilayer
Hydrophobic Lipid Tail
Hydrophilic Head group
Cholesterol
4
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)
5
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?

6
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
7
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.
8
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.
9
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

10
WHATS 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

11
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

12
EXPERIMENTAL CONSIDERATIONS FOR SPT EXPERIMENTS

• TEMPERATURE
• AB COATED BEADS MEET OBSTACLES EVERY 1mM AT 220C
• AB COATED BEADS MEET OBSTACLES EVERY 3-4mM _at_
  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 CANT 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