W4L3 Flashcards
Fluidity of the Membrane
Main things:
- Depends on Level of unsaturated:saturated fatty acids
- Temperature
Other factors:
- Cholesterol content
- Proteins
Saturated at room temp is more solid
Saturated fatty acids found in meat, butter; at room temp it is more solid.
More saturated = more solid = less fluid
more proteins = less fluidity
Unsaturated = more fluid membranes
Too little cholesterol = not enough membrane fluidity
Just the right amount of cholesterol = membrane fluidity
Too much cholesterol = packs together = not enough membrane fluidity
Membrane Asymmetry
In the ER, as you are making phospholipids and exporting them to plasma membrane…the PC, PS and PI are found equally on each side of the ER membrane; also not many glycolipids in ER
As you go to the plasma membrane, there are glycolipids on extracellular portion of PM. There is asymmetry in PC, PS and PI. (serine, choline, inositol)
- Glycolipids on outer membrane of PM
- More PC in outer membrane of PM
- PS in the inner membrane of PM
- PI on endosomes
Lipid Transport within the Cell
- Lateral diffusion
- rapid
- moving within the 2 dimensional bilayer
- the fastest of them all - Vesicular transport
- rapid
- vesicles move the lipids back and forth throughout the cell (ER to PM, endocytosis) - Monomeric exchange
- 2 organelles bumping into each other and exchanging phospholipids, cholesterol, between the 2 membranes - Transbilayer movement
- if this is not catalyzed, this is very very slow
- the cell needs to catalyze this
- enzymes for catalysis are called flippase, floppase, scramblase
- Flippase uses ATP; takes glycerophospholipids from outer leaflet and puts it on inner leaflet; phosphotidylserine is an example of substrate for flippase
- floppase uses ATP; takes phosphotidylcholine (glycerophospholipids) from inner leaflet to outer leaflet
- scramblase randomly takes a glycerophospholipids from one side and brings it to the other side; only calcium dependent
Flippase and floppase active when cell is healthy
When cell undergoes apoptosis, the flippase and floppase lose their activity and are endocytosed. BUT scramblase is always there ready to continue working bc it does not need ATP; exposes phosphotidylserine to the outside of the cell since theres lots of it inside the cell due to inactivity of flippase now; this is signal to immune system that the cell is dying and tells immune system to take care of the cell. the cell continues to dye and immune system aids it
Phosphatidylserine
Changes in membrane asymmetry can lead to disease
PS belongs in the inner membrane
Exposure of PS on the outer leaflet is a recognition signal for apoptosis to proceed
- via the binding of macrophages
- This is normal system
Diseases causing or caused by membrane asymmetry. This is based on phosphatidylserine being on the outside of the cell
- Sickle-cell anemia, thalassemia, kidney stones, malaria, and pre-eclampsia
Membrane rafts = lipid rafts
Small (10-200 nm)
Heterogeneous
Composed of
- Cholesterol
- Sphingolipids
- Phospholipids
- Specialized proteins (caveolin, flotillin, etc.)
More ordered and tightly packed than surrounding bilayer
- due to proteins and cholesterol in lipid rafts
Increased order
Less movement
Thicker lipid bilayer
Buoyant
Detergent resistant
Membrane raft functions
- Trafficking
- can move back and forth from golgi apparatus to PM, and move cholesterol around rapidly
- Involved in trafficking of membrane proteins - Signalling (Compartmentalize cellular processes)
- signalling platform
- if you want proteins to interact, they can all sit in the membrane raft pre-clustered. So if you add a ligand, they are all pre-clustered and ready to signal. As opposed to needing to find many microns across the membrane to find each other
- Organizing centers
– Assembly of signaling molecules
– Signaling can be promoted or dampened - Endocytosis (Concentrate/ separate proteins within the plane of the bilayer)
- if you endocytose via membrane rafts, they usually go to specialized organelles called caveosomes which may or may not feed into the lysosomal system
Effects membrane fluidity
- Lateral diffusion
Also, involved in cell-to-cell junction, in interactions: tight junction and desmosome, but not adherens junction
Composition of membrane raft
- Resident proteins
- Caveolin
- Flotillin
- GPI-anchored proteins (for e.g., prion protein) - Non-Resident Signaling proteins (don’t hang out in the raft) (can move in or out of the lipid raft)
- some G-proteins/ G protein coupled receptors
- non-receptor tyrosine kinases
- etc. - Cytoskeletal/Adhesion proteins
- Actin/ Intermediate filament association
- Cadherins (usually desmosomes)/ Tight Junctions
- Etc.
Also
Increased [Cholesterol] (2x more than non-raft bilayer)
Enriched in proteins containing:
-Glycosylphosphatidylinositol (GPI) anchors
- Longer transmembrane domains
Tight junction, adherens junction and desmosome in membrane raft
- Tight junction
- claudins/occludins
- actin association
- raft associated
- palmitoylation - Adherens junction
- classical cadherins
- actin association
- non-raft associated
- non-palmitoylated
- shorter transmembrane domains - Desmosome
- desmosomal cadherins
- keratin linkage
- raft associated
- palmitoylation
- thicker lipid bilayer
- longer transmembrane domain
Caveolin
Resident protein of membrane raft
If you take a membrane and put lots of cholesterol in there and sphingolipids, then proteins like caveolin and flotillin insert themselves into the membrane. They are not transmembrane proteins though. They will create caveolin scaffolds. Caveolins are sticky and bind to many proteins. Bc of association with other proteins, they can drag other proteins into membrane rafts. Although not as good as clathrin at causing invagination of a membrane for pinching off, they DO deform the membrane somewhat. They make little caves, called caveolae (50 nm across). These are smaller than clathrin-coated pits (100 nm across).
There is caveolin-1 and caveolin-2
Flotillin
Resident protein of membrane raft
Inserts itself into the membrane but does not create a cave like caveolin did; it is just a flat membrane
Called flotillin bc of the way you isolate it. Isolate lipid rafts from non-raft membrane by doing sucrose density ultracentrifugation. You lyse the cells and put them in a high sucrose dense environment. Then overlay it with 35% sucrose, then 10% sucrose. After you centrifuge it 200,000 G overnight for 16 hours, then parts of the lipid rafts will separate from the rest of the membrane and will float to the top of your gradient. Flotillin floated with the lipid raft to the top of the gradient.
2 types of membrane rafts
- Caveolae
- small, flask-shaped invaginations of the plasma membrane
- enriched in caveolin - Planar lipid rafts
- found in neurons
- enriched in flotillin
Caveolin and flotillin can recruit signaling proteins
- proteins can also be present in the brain and interact with nt
- nt to G protein coupled receptor to g protein to effector
Signaling can be promoted or dampened
- bringing an agonist can dampen a signal, for example
Note: receptors, effectors and G proteins do not need to all be in the raft or out of the raft at the beginning of an experiment or analysis. They can be all inside, all outside or some inside some outside. What matters is that all 4 components come together to create the signal. Can occur outside raft or inside the raft.
Rafts can either cluster things, keep them apart. The clustering can occur after agonist, and either inside or outside of the raft
Membrane raft - Disease Processes
- Alzheimer’s disease
- Platforms for production of amyloid-β (neurotoxic protein)
- makes clusters that kill neurons - Prion disease (Creutzfeldt-Jakob disease)
- Normal prion protein (PrPc) is converted to abnormal proteins (PrPsc) in lipid rafts (GPI anchor required)
- Conformational change of normal protein, in beef; normal heat will not kill it
- As prions build up in cells, the brain shrinks and tissues fill with holes
- lose ability to think and move properly, memory loss
- fatal
- occurs via ingestion of prion protein and goes to ur brain
- Prion IS a normal protein we have in the brain, but the PrPsc inserts itself into the membrane. As soon as the lipid rafts come together, PrPsc tells the normal prion protein to change configuration. This is a conformational transfer. The normal protein then becomes PrPsc and the disease spreads
- all this occurs on lipid rafts
Membrane raft challenges
Main challenge in this field of study: limited methods
Difficult to study lipid rafts in intact cells
Lipid rafts are too small to be resolved by light microscopy, needs live cell imaging
Sooooo we do….Manipulation of cholesterol within the membrane to identify the lipid rafts
- Sequestration of cholesterol
- Depletion or removal of cholesterol from the membrane
Cytoskeleton
Network of protein fibers that extend throughout the cytoplasm
Nomenclature is based on size
Many functions:
- Cell adhesion and movement (migration)
- Cell shape and structure
- Endocytosis/exocytosis (secretion and recycling)
- Organelle/ protein transport
- Mitosis/ Cytokinesis
- Cilia and Flagella
- Muscle contraction
- etc.
Cytoskeleton categories
- Microtubules
- Structure: protofilaments
- Diameter: 25 nm
- i.e. Tubulin (a and b subunits) - Actin (microfilaments)
- Structure: double helix
- Diameter: 6-7 nm
- i.e. Actin - Intermediate filaments
- Structure: two anti-parallel helices, form tetramers
- Diameter: 10 nm
- i.e. Vimentin (mesenchymal cells), Desmin (muscle cells), Keratins (epithelial cells), Nuclear Lamins
