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

Lecture 4 Membrane: 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

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

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

Four major phospholipids (50% of lipid)

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

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

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

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

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

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

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.

Glycosylation and disulfide bonds

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

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

Spectrin is a cytoskeletal protein noncovalently associated with 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 Fluorescence recovery after photobleaching Fluorescence Loss in photobleaching

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 Glycoproteins Glycolipids Some proteoglycans Ruthenium red Lectins

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.