Membranes and lipid rafts Flashcards
Plasma membrane
Encloses the entire cell, defines its boundaries, and maintains the essential difference between the cytosol and the extracellular environment. Cell membranes are dynamic, fluid structures, and most of their molecules move about in the plane of the membrane. There are also membranes enclosing organelles in eukaryotic cells
Lipid bilayer
Provides the basic fluid structure of the membrane and serves as a relatively impermeable barrier to the passage of most water-soluble molecules. Most membrane proteins span the lipid bilayer and mediate nearly all of the other functions of the membrane. The lipid bilayer forms due to the special properties of lipid molecules, which assemble spontaneously into bilayers even under simple artificial conditions
Functions of membrane proteins
This includes the transport of specific molecules across it and the catalysis of membrane associated reactions, like ATP synthesis. Some membrane proteins connect the cytoskeleton through the lipid bilayer or to an adjacent cell. Multiple kinds of membrane proteins are necessary to enable a cell to function and interact with its environment
Amphiphilic
Molecules with both hydrophobic and hydrophilic properties. In cell membranes, the lipid molecules have a polar end and a nonpolar end
Types of membrane proteins (2)
- Integral membrane proteins
- Proteins with lipid portions that anchor them in the membrane
Composition of biomembranes
Biological membranes are amphiphilic and are 50% lipid, 50% protein by mass. Phospholipids are the most abundant membrane lipids, and the main phospholipids in most animal cell membranes are phosphoglycerides
Phospholipids
The most abundant membrane lipids. They have a polar head containing a phosphate group and 2 nonpolar hydrocarbon tails (fatty acid tails). 1 usually has 1 or more cis-double bonds (unsaturated) while the other one has no double bonds (saturated). Phosphoglycerides are the main phospholipid in the cell
Phosphoglycerides
The main phospholipid in most animal cell membranes. They have a 3 carbon glycerol backbone.
Phospholipid structure
The polar head group contains 3 compounds- glycerol, phosphate, and choline, with choline being the polar head group and the most external. The head group compound may change between phospholipids. The 2 fatty acid chains are bonded to glycerol. One of the chains contains a double bond and is considered unsaturated. The double bond creates a “kink” in the fatty acid tail and causes this lipid to pack more loosely with other lipids. The saturated chain is straight and rigid, and packs a lot tighter with other lipids. The fatty acid tails are linked to the carbon atoms of glycerol through ester bonds
Saturated
A lipid molecule without double bonds. This molecule will therefore be saturated with as many hydrogens as it can be bound to. Saturated lipids are able to pack more tightly
How does structure change between different membrane phospholipids?
Besides the head group, the length and saturation of the fatty acids may change. Otherwise, the structure stays relatively consistent between different phosphoglycerides
Main mammalian phosphoglycerides (3)
- Phosphatidylethanolamine
- Phosphatidylserine
- Phosphatidylcholine
All of these phosphoglycerides are named for the head group they contain
Ethanolamine
Primary amine/alcohol, acts as a head group for phosphoglycerides
Serine
An amino acid that acts as a head group for phosphoglycerides
Choline
A nutrient within B complex vitamins which acts as a head group for phosphoglycerides
Bacterial phosphoglycerides (2)
- Phosphatidylglycerol
- Cardiolipin- a modified version of phosphatidylglycerol
The 3 mammalian phosphoglycerides generally are not found in bacterial membranes
Phosphatidylglycerol
Contains the molecule glycerol as its head group, found in the bacterial cell membrane. Glycerol also makes up the backbone of these molecules
Cardiolipin
Almost like a double version of phosphatidylglycerol, except for the head group, which is just one glycerol molecule. The molecules also differ in their fatty acid tail length and in the degree of saturation in their fatty acid tails. Cardiolipin actually does exist in eukaryotic cells, as part of the inner mitochondrial membrane. This is one of the lines of evidence for endosymbiotic theory
Phospholipids that are NOT phosphoglycerides (2)
- Sphingomyelin
- Sphingosine
Sphingomyelin
A phospholipid that is not classified as a phosphoglyceride as it does not have a glycerol backbone. Its backbone is composed of sphingosine (which contains a fatty acid tail as part of its backbone). One of its fatty acid tails is attached to the amino group of sphingosine, creating an intermediate called ceramide. Once ceramide acquires its head group, it becomes mature sphingomyelin. The head group, phosphocholine, is attached to the terminal hydroxyl. The fatty acid tails of sphingomyelin have a much higher degree of saturation than the fatty acid tails of phosphoglycerides
Sphingosine
A phospholipid that forms the backbone of sphingomyelin. It is a long acyl (fatty acid) chain with an amino group (NH3) and 2 hydroxyls
Glycolipids structure
A general term referring to lipids containing a sugar component. They are built from sphingosine, so they have more in common with sphingomyelin than phosphoglycerides. The most complex glycolipids are gangliosides, which were originally found in the nervous system but are now known to be present in many different cells. They self associate through hydrogen bonds in sugars, and there are van der waals forces between tails
Location of glycolipids
Glycolipids are exclusive to the outer leaflet of the membrane and allow for sugar molecules to be found on the surface of the cell- this is called the glycocalyx. They are saturated, tight packing, ordered lipids, and therefore are found in the ordered regions of membranes. Glycolipids are found in all animal cell membranes and make up about 5% of the outer leaflet.
Gangliosides
The most complex glycolipids. They are oligosaccharides with 1 or more sialic acids, which are negatively charged. They are found in many different cells, but are abundant in neurons.
3 examples of glycolipids
- Galactocerebroside
- GM1 ganglioside
- Sialic acid (NANA)
Membrane asymmetry
There is an asymmetry to the distribution of lipids in the membrane. For example, glycolipids are generally only found in the outer leaflet of the membrane. The inner half of the membrane is the only place where you will find phosphatidylserine (negatively charged) in a healthy cell. Phosphatidylinositol (phosphoinosites) is also only found in the inner leaflet of the membrane
Phosphatidylserine externalization
PS may be found in the outer half of the cell during apoptosis. It is externalized through the actions of an enzyme called scramblase. This process only occurs in dying apoptotic cells
Phosphoinositides asymmetry
PI is a phospholipid that is exclusive to the inner leaflet of the membrane. They can be phosphorylated in different positions and combinations. These lipids serve as binding sites for other molecules in the cell, usually proteins
Cholesterol
A specialized lipid, but not a phospholipid. Eukaryotic plasma membranes contain large amounts of cholesterol, with there being 1 cholesterol molecule for every phospholipid. Cholesterol is a tight packing lipid due to its fully saturated hydrocarbon tail and rigid ring structure.
Cholesterol structure
It has a fully saturated nonpolar hydrocarbon tail attached to a steroid ring structure. The ring structure is the rigid part of the molecule, and an OH group is attached to it, which serves as a small polar head group
Orientation of cholesterol
Cholesterol orients itself in the membrane with their hydroxyl group close to the polar head groups of adjacent phospholipid molecules.
Function of cholesterol
When cholesterol is inserted between 2 neighboring phospholipids, it will help them to pack a little tighter. It acts like a molecular glue between phospholipids.
Hydrophobicity
The property of nonpolar molecules in water. When put into an aqueous solution, lipids will spontaneously aggregate or organize into micelles or bilayers. These aggregates are energetically favorable because the lipids are orienting their nonpolar (hydrophobic) portions to limit their contact with water. This also serves as a natural “healing” property
Micelles
Micelles are natural formed by cone shaped amphiphilic molecules. The lipids are coned shaped because they only have one fatty acid tail. In aqueous solution, the fatty acid tails are oriented inward, creating a spherical structure with the fatty acid cone tips pointed toward the center. The interior of the micelle is completely hydrophobic. Micelles are important in detergent and soap solutions
Bilayers
Cylinder shaped amphiphilic molecules, like phospholipids, spontaneously form bilayers in aqueous solution. The fatty acid tails of the lipids point inward to limit their contact with the water
What happens when a polar molecule is placed in water?
Polar molecules contain one charge at one end of the molecule and an opposite charge on the other end. In water, the oxygen contains a partial negative charge and the hydrogens contain a partial positive charge. Hydrogen bonds can form between water molecules or between water and a polar molecule. The electronegative oxygen pulls electrons away from hydrogen, which then forms a hydrogen bond with another oxygen molecule. The hydrogen bonding property allows polar molecules to be soluble in water, hence why they are called hydrophilic molecules.
What happens when a nonpolar molecule is placed in water?
Nonpolar molecules lack charge and therefore cannot form hydrogen bonds with water molecules. The water molecules form a cage-like arrangement around the nonpolar molecule
Why are fatty acids nonpolar?
There is no separation of charge because carbon and hydrogen are too close together in electronegativity and share electrons equally
Why must bilayers form cell-like enclosures?
The hydrophobic effect forces bilayers to form a round shape. A flat/planar bilayer is energetically unfavorable because there is a free hydrophobic edge of the bilayer in contact with the aqueous solution. Once the bilayer forms a sealed compartment, the hydrophobic areas have minimal contact with water
Cortical cytoskeleton
Directly underlies and has connections to the membrane proteins- located in the cytoplasm. It is mostly composed of actin. The connections to the membrane allow the membrane to be anchored to the cytoskeleton. The cortical cytoskeleton is also responsible for weird-looking cells. Cells with microvilli have that structure due to the arrangement of actin
Properties of membrane bilayers (4)
- 2-D fluidity
- Lipid rotation
- Phase transition
- Membrane phases
2-D fluidity of membranes
There can be lateral motion/diffusion of lipids or protein within the 3D structure. The molecules are able to turn on their axis. There is no vertical motion. However, flip-flop of the molecules won’t typically occur- this is a controlled process that requires enzymes
Lipid rotation in membranes
Depends on the temperature and composition of the membrane. A lower temperature will be less fluid and a higher temperature would be more fluid. In terms of composition, a membrane with more saturated lipids will be less fluid and a membrane with more unsaturated lipids will be more fluid.
Membrane bilayer phases
- Gel
- Liquid disordered (LD)
- Liquid ordered (LO)
Membrane phase transition
A synthetic bilayer made from single type of phospholipid changes from a liquid state to a two dimensional gel state at a characteristic temperature. This change of state is called a phase transition. The temperature at which it occurs is lower (the membrane becomes more difficult to freeze) if the hydrocarbon chains are short or if they have double bonds. A shorter chain length reduces the tendency of the hydrocarbon tails to interact with each other
Gel phase
Occurs at lipid freezing point, doesn’t really exist in nature or at physiological temperatures. At this point, the membrane has virtually no fluidity and there is no molecular motion of lipids.
Liquid disordered and liquid ordered membrane phases
Both of these phases exist at physiological temperatures, and both can exist in the same membrane at the same time. Parts can be liquid ordered and other parts can be liquid disordered. LD is more fluid and allows for more molecule motion and lateral mobility. This portion of the membrane is likely to be composed of more unsaturated lipids. LO is composed of saturated lipids (cholesterol is an important molecule here). It is less fluid and rigid
How does cholesterol influence membrane permeability?
When cholesterol is mixed with phospholipids, it enhances the permeability and barrier properties of the lipid bilayer. Cholesterol’s rigid steroid rings interact with and partly immobilize the regions of hydrocarbon chains that are closest to the polar head groups. By decreasing the mobility of the first few hydrocarbon groups of the chains of the phospholipid molecules, cholesterol makes the lipid bilayer less deformable in this region and therefore decreases the the permeability of the bilayer to small water soluble molecules. Although cholesterol tightens the packing of the lipids in a bilayer, it does not make membranes any less fluid. In eukaryotic plasma membranes, cholesterol prevents the hydrocarbon chains from coming together and crystallizing.
Fluid-mosaic model
An old model proposing that the membrane is made up of a homogenous mixture of lipids and proteins
Lipid rafts
Regions of the membrane that exist in an ordered (LO) state. Ordered “rafts” float (move) freely across the LD “sea” of unsaturated lipids. These structures can be long standing or short lived. Specific membrane proteins and lipids are seen to concentrate in a more temporary, dynamic fashion facilitated by protein-protein interactions that allow the transient formation of specialized membrane regions. The tendency of mixtures of lipids to undergo phase partitioning, as seen in artificial bilayers, may hep to create rafts in living cell membranes
Composition of lipid rafts
Since these are ordered regions of the membrane, they are rich in sphingolipids like sphingomyelin, and are rich in cholesterol. A high degree of saturation in sphingolipids and the rigidity of cholesterol is responsible for this structure, cholesterol especially is necessary for a lipid raft to form. There may be other saturated lipids like glycolipids, and at times may contain ceramide. However, sphingolipids and cholesterol are the main components in animal cells. Cholesterol may act as “glue” between sphingomyelin molecules in the rafts, making them pack even tighter
Properties of lipid rafts (6)
- Lipid rafts are thicker regions of the membrane compared to unsaturated regions
- A lot of proteins are found in lipid raft regions, a lot of these are lipid anchored proteins (like GPI anchored proteins)
- An LO to LD phase transition of lipid rafts is up to 15 degrees Celsius above the transition temperature of the surrounding membrane- phase transition of lipid rafts is possible but slightly more difficult
- Cholesterol favors intercalation in between lipids that make up lipid rafts
- Rafts contain a high degree of fatty acid saturation
- Similarity in structure of hydrophobic domains of raft lipids & dissimilarity w/ surrounding fluid phospholipids favors self-association, which minimizes free energy
Liposome
An artificial membrane made in a laboratory. In one important experiment, the lipids making up the liposome are fluorescently labeled with red fluorescence. One liposome was made from a 1:1 ratio of phosphatidylcholine (PC) and sphingomyelin. This membrane was solid red. Another liposome was made from a 1:1:1 ratio of PC, sphingomyelin, and cholesterol. Once cholesterol was added, there were distinct red regions of the membrane in a “sea” of black. This experiment demonstrates the importance of cholesterol in lipid rafts.
GPI anchored proteins in lipid rafts
These proteins are made as soluble proteins in the cytosol and are subsequently anchored to the membrane by the covalent attachment of the lipid group. The hydrogen bonding of glycosphingolipids with GPI anchored proteins stabilize the complex. The fatty acid tail of these proteins adds saturation in the lipid rafts. This causes the rafts to self associate, as saturated lipids are more similar to each other than they are to unsaturated lipids
Lipid raft functions (3)
- Concentration of signaling receptors and proteins- main function
- Delivery of raft proteins in vesicles
- Role in physical formation of vesicles
Signaling receptors and proteins in lipid rafts
Lipid rafts allow concentration of signaling receptors in a large patch, like a signaling platform, amplifying signaling to a great degree. This induces cell signaling, as signaling proteins are often activated by clustering the proteins together. Some of these proteins are resident to rafts, others are LD proteins that are recruited to the raft. Lipid rafts will coalesce and bring the signaling proteins together. This function exists in the outer and inner leaflet
Ceramide in lipid rafts
Ceramide (a sphingomyelin intermediate) is the strongest raft promoting lipid, but it is not always present. Ceramide tends to be present in the membrane during cell death processes. It helps to strengthen or enlarge lipid rafts. Ceramide helps the rafts to further aggregate and may amplify signaling even more
SMase
When ceramide does form, it will do so through the processing of sphingomyelin, with an enzyme called sphingomyelinase (SMase). The enzyme removes the choline head group from sphingomyelin, creating ceramide
Delivery of raft proteins in vesicles
Lipid rafts can aggregate together and form vesicles, and the vesicles will then bud off the membrane. This can be important for delivering signals (microvesicles, exosomes) to neighboring cells. Lipid rafts also help to form intracellular vesicles in the cytoplasm. These vesicles help with intracellular vesicle trafficking to/from the Golgi. Finally, lipid rafts may contribute to enveloped viruses
Role of lipid rafts in the physical formation of vesicles
The lipids create line tension between the thick lipid raft regions and the thin non-raft regions. Line tension causes curvature in the membrane and results in part of the membrane budding off into a vesicle. Once a vesicle forms, it will pinch off of the membrane
Which cells are lipid rafts present in?
Lipid rafts have been well studied in eukaryotic cells and are generally assumed to exist in all eukaryotic cells. Since most prokaryotes lack sterols, they were thought not to have lipid rafts. However, we know that some prokaryotes have sterols and 1 prokaryote (Borrelia) so far has been shown to have lipid rafts. The Borrelia membrane is structurally different from a typical Gram negative membrane and is therefore more similar to a eukaryotic membrane
Methyl-β-cyclodextrin
One easy method used to study lipid rafts- it specifically removes cholesterol from membranes. Methyl-β-cyclodextrin is made of sugar molecules bound together in a ring. The ring structure basically extracts cholesterol out of the membrane and solubilizes it, and it easily accommodates bulky cholesterol. Interior of ring is not completely hydrophobic, but less hydrophilic than aqueous environment. Since cholesterol has been extracted, it disrupts lipid rafts. Methyl-β-cyclodextrin is also used to make cholesterol free foods
Methyl-β-cyclodextrin structure
Contains a ring of sugar molecules with a central pore. Cholesterol binds in the central pore. There are 3 types of cyclodextrin- alpha, beta, and gamma. All cyclodextrins form a ring structure, but they differ in the size of the central pore (alpha is the smallest, gamma is the biggest). Cholesterol is best accommodated by the beta molecule
Fluorescence anisotropy
Measures the degree of order in a membrane based on the scattering of UV fluorescence. If there is more scattering, the membrane is less ordered and there is a lot of molecular motion. If there is less scattering, the membrane is more ordered
Detergent-resistant membrane (DRM) isolation
Due to their packing, lipid rafts & their associated proteins resist solubilization by detergents, specifically a detergent called Triton-X-100 at 4 degrees Celsius. If Triton-X-100 is added at this temperature, all of the liquid disordered regions would be solubilized, but the liquid ordered regions would not be. Detergent resistant membranes correlate to lipid rafts, and they can be purified and analyzed in density gradients. This method helps to isolate the rafts and can also be used as a measure of raft size
Density gradient centrifugation
In a density gradient, lipid rafts are placed at the bottom of a tube and put through a centrifuge. After the centrifuge, the lipid rafts will migrate to the area in the tube that is consistent with their density. The detergent-solubilized material has migrated to the bottom of the tube. This is because lipid rafts are more buoyant than the cellular material that has been solubilized. This method helps to purify lipid rafts
Forster resonance energy transfer (FRET)
Allows you to determine whether lipid rafts exist in a membrane and identify liquid ordered from liquid disordered domains. Uses 2 fluorescent probes- raft specific probes for saturated lipids and non-raft probes for unsaturated lipids. Looks for fluorescence transfer (transfer of fluorescent energy) to measure if the probes are a certain distance away from each other. The distance is interpreted as a separation of domains
