Summary

1. Cell doctrine;2. Two major types of microscopes: light and electron;3. Limitation of resolution: wavelength of radiation;4. Advantage and disadvantage of light and electron MS5. Different types of light microscopes: bright field,

phase contrast, DIC, dark field,fluorescent, confocal6. Image processing: digital enhancement7. Two major types of EM: TEM and SEM 8. Additional tricks: shadowing, freeze-fracture, freeze

etching, negative staining, tomography;9. Live imaging, calcium indicators, caged compounds,

GFP, pulse chasing

Lecture 4Membrane: lipids and membrane proteins

Plasma membrane

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

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

Closed lipid bilayer membranes are energetically favorable

Diffuse across 2 um in 1 sec!

Needs phospholipidtranslocators

Properties of the lipid bilayerLiposome and black membranes

Cholesterol decreases permeability and fluidity at highConcentrations but also inhibits phase transition

“freeze” or “gel”

Four major phospholipids (50% of lipid)

Lipid rafts are small, specialized areas in membraneswhere some lipids (primarily sphingolipids and cholesterol)

and proteins are concentrated

GPI-linked

70 nm in diameter

The asymmetrical distribution of phopholipids and glycolipids in the lipid bilayer

Phosphotidylcholine and sphingomyelin: outerPhosphotidylethanolamine and phosphotidylserine: inner

Negative inside

PKC requires phosphotidylserine

Apoptotic cells have their phosphotidylserine translocated to outer bilayer

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

Glycolipids are only present on the outer monolayer

ProtectionElectrical effectsCell-recognitionEntry points for bacteria

No genetic evidence

Ganglioside:

Various ways membrane proteins associate withthe lipid bilayer

Glycosylphosphatidylinositol(GPI) and phosphatidylinositol-specific Phospholipase C (PIPC)

Most transmembrane domains are made of helices

Hydrogenbonds inthe polypeptidechain

Some barrels form large transmembrane channels

10 aa is sufficient, barrels are rigidand 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.

Glycosylation and disulfide bonds

Membrane proteins can be solubilized and purified in detergentsamphipathic

Charged (ionic) -sodium dodecyl sulfate (SDS)Uncharged(nonionic) -Triton

The biconcave shape of red blood cells

No nucleus:pure plasmamembrane!

Spectrin is a cytoskeletal protein noncovalently associatedwith the cytosolic side of the red blood cell membrane

Ankyrin connects spectrin to Band 3

Band 4.1 binds to spectrin, actin and glycoporin

Membrane proteins diffuse in the membrane

Measuring the rate of lateral diffusion by photobleaching

Fluorescencerecovery afterphotobleaching

FluorescenceLoss inphotobleaching

Domains of an epithelial cell

Proteins and lipids are NOT always free to go anywhere they want

Intercellular junctions

Membrane domains created in a single cell

The cell surface is coated with a carbohydrate layer

GlycoproteinsGlycolipidsSome proteoglycans

Ruthenium redLectins

Summary

1. Membranes are made of lipids and proteins;2. Lipid bilayer is a energetically favored structure;3. Fluidity, permeability and asymmetry of lipid bilayer4. Membrane proteins and transmembrane domains;5. Membrane protein modifications and asymmetry;6. RBCs are good models for plasma membrane;7. Membrane proteins in some membranes are more free to

move laterally;8. Carbohydrate layer.