1. Summary
Cell doctrine;
Two major types of microscopes: light and electron;
Limitation of resolution: wavelength of radiation;
Advantage and disadvantage of light and electron MS
Different types of light microscopes: bright field,
phase contrast, DIC, dark field,fluorescent, confocal
Image processing: digital enhancement
Two major types of EM: TEM and SEM
Additional tricks: shadowing, freeze-fracture, freeze
etching, negative staining, tomography;
Live imaging, calcium indicators, caged compounds,
GFP, pulse chasing

2. Lecture 4 Membrane: lipids and membrane proteins
Plasma membrane

3. Cell membrane: thin layer of dynamic and fluid lipid and protein
molecules held together by noncovalent interactions
30% of the proteins encoded in our genome are membrane proteins

4. Membrane lipids, like the most abundant kind, phospholipids,
are amphipathic

5. Closed lipid bilayer membranes are energetically favorable

6. Properties of the lipid bilayer
Liposome and black membranes
Diffuse across
2 um in 1 sec!
Needs phospholipid
translocators

7. Cholesterol decreases permeability and fluidity at high
Concentrations but also inhibits phase transition
“freeze” or “gel”

8. Four major phospholipids (50% of lipid)

9. Lipid rafts are small, specialized areas in membranes
where some lipids (primarily sphingolipids and cholesterol)
and proteins are concentrated
GPI-linked
70 nm in diameter

10. The asymmetrical distribution of phopholipids
and glycolipids in the lipid bilayer
Negative inside
Phosphotidylcholine and sphingomyelin: outer
Phosphotidylethanolamine and phosphotidylserine: inner
PKC requires phosphotidylserine
Apoptotic cells have their phosphotidylserine
translocated to outer bilayer

11. Phosphotidylinositol--
lipid kinases add phosphate groups in the inositol ring (PI3-kinase)
Phospholipases cleave the inositol phospholipid molecules into two
fragments

12. Glycolipids are only present on the outer monolayer
Ganglioside:
Protection
Electrical effects
Cell-recognition
Entry points for bacteria
No genetic evidence

13. Various ways membrane proteins associate with the lipid bilayer
Glycosylphosphatidylinositol(GPI) and
phosphatidylinositol-specific Phospholipase C (PIPC)

14. Most transmembrane domains are made of  helices
Hydrogen
bonds in
the polypeptide
chain

15. Some barrels form large transmembrane channels
10 aa is sufficient, barrels are rigid
and hyropathy plots cannot identify barrels
Crystallize readily, abundant in outer membranes of mito, chloroplast
and bacteria. Pore-forming proteins (porin). Loops of polypeptide chain in the lumen.
Selective (maltoporin). Some are receptors or enzymes not transporters.

16. Glycosylation and disulfide bonds

17. Membrane proteins can be solubilized and purified in detergents
amphipathic
Charged (ionic) -sodium dodecyl sulfate (SDS)
Uncharged(nonionic) -Triton

19. The biconcave shape of red blood cells
No nucleus:pure plasma
membrane!

20. Spectrin is a cytoskeletal protein noncovalently associated
with the cytosolic side of the red blood cell membrane

21. Ankyrin connects spectrin to Band 3
Band 4.1 binds to spectrin, actin and glycoporin

22. Membrane proteins diffuse in the membrane

23. Measuring the rate of lateral diffusion by photobleaching
Fluorescence
recovery after
photobleaching
Fluorescence
Loss in
photobleaching

24. Domains of an epithelial cell
Proteins and lipids are NOT always free to go anywhere they want
Intercellular junctions

25. Membrane domains created in a single cell

26. The cell surface is coated with a carbohydrate layer
Glycoproteins
Glycolipids
Some proteoglycans
Ruthenium red
Lectins

27. Summary
Membranes are made of lipids and proteins;
Lipid bilayer is a energetically favored structure;
Fluidity, permeability and asymmetry of lipid bilayer
Membrane proteins and transmembrane domains;
Membrane protein modifications and asymmetry;
RBCs are good models for plasma membrane;
Membrane proteins in some membranes are more free to
move laterally;
8. Carbohydrate layer.