Biology 1020 Unit 2

Amphipathic

hydrophilic component and hydrophobic component in same molecule

Cholesterol

small hydrophilic head, steroid rings (more rigid), hydrocarbon tail (amphipathic molecule)

how do phospholipids arrange themselves?

with their hydrophobic tails touching on the inside (away from aqueous environment) and hydrophilic heads at the top and bottom (towards extracellular space or cytoplasm respectively)

liposome

tiny, synthetic, spherical vesicles composed of a lipid bilayer—similar to cell membranes—that can encapsulate drugs. they are built in vitro and can have membrane proteins inserted

endocytosis

an active cellular transport process that engulfs extracellular materials—fluids, nutrients, or pathogens—by merging off portions the plasma membrane to form internal vesicles.

exocytosis

intracellular vesicles fuse with the plasma membrane, releasing their contents (neurotransmitters, hormones, or waste) into the extracellular space

lumen

internal volume enclosed by a membrane

membrane function

1) compartmentalization: forms a physical boundary that divides cellular space for different purposes

2) scaffold for biochemical activities: interacting cellular components can be localized to membrane (embedded or peripherally-attached) for more effective interaction

3) selectively permeable barrier: molecules require gate or channel to enter cell; protects cell

4) signal transduction: membranes have receptors that combine w/ specific molecules (ligands) to regulate processes in cell

5) interaction w/ neighboring cells or ECM

lipid bilayer/plasma membrane composition

1) amphipathic membrane lipids (phosphoglycerides, glycolipids, cholesterol)

2) membrane proteins: integral proteins, peripheral, lipid-anchored —> proteins are all free to move within the plane of the membrane unless they are bound

3) membrane carbohydrates in OUTER leaflet: glycoproteins or glycolipids

4) lipid rafts: membrane domains concentrated in cholesterol and lipids with long saturated tails

integram membrane/transmembrane proteins

penetrate lipid bilayer; have domains that protrude both sides of the membrane

• their membrane spanning hydrophobic alpha helices tend to be 20-25 amino acids in length

• can be used for signaling: receive ligands on extracellular side and relay info to intracellular side

• can also be channels or transporters

• are amphipathic (the part that spans membrane is hydrophobic, the rest is hydrophilic)

peripheral membrane proteins

located entirely outside of lipid bilayer, associated by noncovalent bonds

• can be extracted w/o disrupting the membrane (using high/low salt or pH)

• can be recruited and released as needed

lipid-anchored membrane proteins

located outside lipid bilayer but are covalently linked to a lipid molecule in the bilayer

• ie. GPI anchored protein

where are membrane carbohydrates located

on the outer leaflet; they point into extracellular space. cells are “sugar-coated“. sugars may be attached to lipid or protein (glycolipid or glycoprotein)

glycoproteins

oligosaccharides are attached to amino acids of a membrane protein

glycolipids

oligosaccharides are attached to membrane lipids

what is the fluid mosaic model of the plasma membrane made of

• membrane lipids (phospholipids, cholesterol, etc), proteins (integral membrane, peripheral, lipid-linked), carbohydrates (glycoproteins, glycolipids)

• very flexible and dynamic fluid

cholesterol function

• cholesterol fits easily into gaps between phospholipid molecules in a lipid bilayer

• physically interrupts the van der Waals interactions between nonpolar tails —> helps make the membrane more fluid in colder temperatures

• cholesterol also has rigid steroid rings, so they could also provide structure/rigidity in warmer temperatures

• overall, it acts as a buffer to keep the membrane within the optimal fluidity range for a wider range of temperatures and helps the membrane stay stable

lipid rafts

• specialized membrane regions where there are certain types of lipids that interact favorable with each other and travel toegther

• example of lateral asymmetry since they are stiffer/more rigid regions of cell membrane containing more cholesterol and saturated fatty acid tails —> tighter packing (different from the rest of the surrounding cell membrane environment aka a sea of disordered phosphatidylcholine)

• high concentration of signaling proteins and molecules, centers for signal transduction

lateral asymmetry

asymmetry looking at just one leaflet (regions of the same leafet having different properties)

• ie. lipid rafts

• tight junction-mediated segregation of outer leaflet lipids of the apical and basolateral sides of epithelial cells

• sound sensing hair cells of the inner ear

transbilayer asymmetry

refers to difference between inner and outer leaflet. the two different leaflets have distinctly different lipid compositions

how do the outer and inner leaflets differ in composition?

• outer leaflet is rich in phosphatidylcholine (PC) and sphingomyein (localizes to lipid rafts). Glycolipids are only found in this leaflet for cell signaling purposes.

• phosphatidylserine (PS) which is negatively charged is only on inner leaflet —> charge asymmetry

• phosphatydlinositide is also confined to the inner leaflet, which is a key signaling lipid and protein docking lipids

Flippase enzymes move inner membrane lipids inside if they accidentally flip over to maintain membrane asymmetry

Overall, cholesterol is evenly distributed between leaflets, but the phosphoglycerides (PC, PS, PE, and PI) and sphingomyelin (SM) are asymmetrically distributed

key experiments that proven existence of fluid mosaic model and lipid bilayer

• Gorter and Grendel: measured the lipid content/cell surface area of red blood cells —> supports lipid bilayer

• FRAP: shows recovery after photobleaching (showed membrane fluidity)

• Freeze fracture TEM: showed that proteins penetrate through membranes —> supports fluid mosaic model

• Frye and Edidin: demonstrates rapid intermixing of human + mouse antigens —> shows membrane fluidity

Gorter and Grendel experiment (1925)

1. isolate RBCs from whole blood by using low speed centrifugation to pellet RBCs and leave white blood cells as a layer on top of the RBC pellet

2. calculated surface area of red blood cell (good choice because it has no internal organelles —> no extra intracellular membranes)

3. extract lipids from known number of RBCs using organic solvent

4. forms a lipid monolayer on water trough (phospholipids spontaneously arrange themselves so that their hydrophobic heads are in the water)

5. measure surface area of monolayer

the surface area of monolayer was double the surface area of the RBC determined by light microscopy —> showed that there was a bilayer

Iateral diffusion

membrane phospholipids move rather quickly within respective leaflets spontaneously

transverse flip flop

requires flippase enzyme… phospholipids do not spontaneously move from one monolayer to another

what does membrane fluidity depend on?

• length of hydrocarbon tails: longer tails —> more VDWs —> tighter packing —> less mobility

  • shorter tail membranes are more fluid at cooler temperature

• temperature: higher temperatures —> more fluid bc phospholipids can make and break VDWs more easily

• cholesterol: has rigid ring structure, so it helps maintain fluidity @ optimal level at high and low temperatures; acts as a stabilizing buffer

• ratio of unsaturated fatty acid tails to saturated fatty acid tails:

  • unsaturated phospholipids have kinks & more double bonds —> not able to pack as closely —> less VDWs —> more fluid (lower melting point)

  • saturated phospholipids are straight chains —> tight packing —> more VDWs —> less fluid

  • all cell membranes contain mix of unsat and sat fatty acids… its the ratio that determines fluidity

transition temperature

the specific temperature at which a biological membrane changes its physical state from a rigid, ordered "gel" phase to a fluid, disordered "liquid-crystalline" phase. as temperature increases, there is a sharp transition between rigid and fluid states. the transition temperature is this temperature

what is the importance of fatty acid unsaturation for membrane fluidity? of enzymes that desaturate fatty acids?

• unsaturated fatty acids allow for more membrane fluidity. more double bonds —> more kinks —> less lateral packing —> less VDWs —> more fluidity

what is meant by the membrane’s transition temp, and how is it affected by the degree of saturation or length of fatty acyl chain? how are these properties important in the formation of lipid rafts.

• the membrane’s transition temp is the temp at which it goes from solid/rigid to fluid state.

• If there is a greater proportion of unsat, fluidity will be higher since less tight packing —> less VDWs.

• The shorter the fatty acid tails are, the more fluid the membranes since there is less space for VDWs and interactions.

• Lipid rafts would have longer fatty acid tails and more saturated phospholipids since lipid rafts are stiffer and thicker

how can the two sides of a lipid bilayer have different ionic charges? what is the functional significance of this?

different ionic charges occur bc the two sides are composed of different phospholipids with different properties. Phosphotidylserine (negatively charged) is located solely on the inner leaflet by flippase. This is important since many signaling proteins in cytosol are positively charged, so they attract. Additionally, difference in charges creates an electrical gradient across the membrane to store energy

Fluorescence recovery after photobleaching (FRAP)

method for measuring fluidity of membrane or mobility of membrane protein (lateral diffusion)

FRAP process

1. label membrane lipids or proteins w/ fluorescence (tag w/ antibodies and very small fluorophores)

2. photobleach spot with laser beam

3. measure rate of recovery in that region over time… membrane lipid or protein should spontaneously diffuse back into “blank“ area if they are mobile

4. rate of recovery can tell you how fluid it is

does the cell need to be fixed for labeling a membrane lipid

no, we’re labeling something on the membrane and don’t need to get inside cell. we don’t have to poke holes in the membrane.

RE: we always have to fix cells for immunofluorescence since we’re tagging a protein/molecule inside the cell

photobleaching

high energy dose of blue light (for fluoroscein) or green light (for rhodamine) induces covalent changes in ring structure, destroying capacity to emit additional photons of green/red light

rhodamine

fluorophore that is excited by green light and emits red light

fluorescein

fluorophore that is excited by blue light and emits green light

FRAP graph for a fully mobile lipid/protein

graph shows full recovery at the same level as before

FRAP graph for bound lipid/protein

not full recovery, graph plateaus and does not reach original height

how do we label integral membrane proteins for FRAP?

1. direct immunofluoresence: use a primary antibody that has a fluorophore conjugated directly to it (no secondary antibody bc we want the tag to be as small as possible)

2. transfect cells with construct (DNA plasmid) encoding GFP-tagged membrane protein (fusion protein)

why would fluorescence not recover after FRAP?

protein’s lateral mobility may be restricted or anchored down in some way

modes of restricting integral membrane protein lateral mobility

• lateral clustering of IMPs (more closely packed)

• ECM binding/clustering of IMP: anchored to something in ECM

• cytoskeletal restraint of IMPs: anchored to something in cytoplasm (interior)

  • usually a scaffold protein (ankyrin or spectrin)

• cell adhesion mediated clustering of receptors (cell-to-cell interaction)

  • ie. tight junctions

gap junction

communication portal/tunnel between two neighboring cells; made up of connexions (transmembrane protein). not mobile at all because they are physically bound together (in FRAP, the photobleached gap does not close). allows small ions, sugars, amino acids, etc to flow directly from one cell to another

lateral aggregation

occurs within the plane of a membrane; proteins cluster together and move slower through the membrane (ie. lipid raft). for a gap junction, the connexons huddle together in a dense patch in the membrane

extracellular aggregation

they are completely stuck and immobile bc they are physically bound to connexons of the other cell in a gap junction. occurs when proteins are forced into a cluster by something outside the cell.

freeze fracture TEM

1. freeze tissue in liquid nitrogen chilled solvent

2. specimen is split open w/ sharp blade (chooses path of least resistance and splits down the bilayer)

3. peel membrane layer back and deposit heavy metal stain on newly exposed surfaces

4. TEM. Lots of bumps will be visible, while opposite leaflet has pit where the protein was displaced

P face: cytoplasm side, E face: extracellular side

Frye and Edidin experiment (1970)

1. fused a mouse cell and human cell into a heterokaryon

2. labeled mouse antigens (fluorescein) on one side and human antigens (rhodamine) on the other side (secondary antigens)

3. they saw rapid intermixing of the antigens within 40 minutes, demonstrating that the membrane is fluid

also showed that mosaic formation is dependent on temperature: warmer temps = more mosaic

lateral asymmetry in sound sensing hair cells of inner ear

FRAP demonstrates that the apical side has a higher recovery than lateral and basal sides —> apical side is more fluid. tight junctions act as physical barricade and prevents lipids and proteins from apical area from drifing down into basal domain. need to maintain lateral asymmetry

epithelial cells and lateral asymmetry

• apical side is tougher & made to face harsh conditions of lumen

  • more saturated fatty acids, cholesterol, and lipid rafts

• basal lateral side is less tough

• maintained by tight junctions

tight junctions

composed of transmembrane proteins that reach out from the cell and bind directly to some proteins on neighboring cell

• fuses two outer leaflets of two membranes together

  • can serve as a zipper to prevent leakage (ie. prevents acid from gut from leaking into bloodstream)

  • can serve as a dam to prevent contents from apical side and basolateral side from mixing

• restricts lateral mobility of outer but not inner leaflet

active transport

energy is required to move this solute up or against its concentration gradient from low to high concentrations. can occur directly with ATP hydrolysis or indirectly (cotransport) without ATP

what type of molecules can cross the membrane through simple diffusion?

• small nonpolar molecules (ie. O2)

• small uncharged polar molecules (H2O) (can also use aquaporin channels for bulk movement)

what types of molecules cannot cross the membrane by itself?

• ions

• large polar molecules

structural anatomy of the bacterial potassium (K+) ion channel

• contains selectivity filter that only allows dehydrated K+ ions to pass through channel (no larger ions or smaller ions (giving up hydration shell is unfavorable))

• K+ is positively charged, and the selectivity filter contains electronegative oxygens which are partially negative —> draws K+ in

• contains gating alpha helices, which decide when the ions can pass (must go through conformation change)

types of ion channel gates and how they open

• voltage gated: responds to an initial stimulus (slight voltage change and depolarization) —> triggers opening of voltage gated ion channel once at threshold —> more depolarization

• ligand gated (can be extracellular or intracellular) —> produces conformational change

• mechanically gated: responds to tension and pressure, usually attached to cytoplasm and is physically pulled open

ligand

any molecule that binds to a receptor

agonist

specific type of ligand that activates the receptor and triggers its normal repsonse. all agonists are ligands, but not all ligands are agonists

voltage gated sodium ion channel opening process

• resting membrane potential is -70mV (inside is 70mV more negative than outside)

• small depolarization occurs —> membrane becomes less negative & reaches threshold ~55mV

• voltage gated Na+ channels open in response, causing a spike and driving action potential as Na+ moves into cell

• repolarization and return to resting state as K+ channel opens and K+ moves out of cell

patch clamp technique

key experiment that allowed scientists to record an ion channel opening and closing

• tiny pipette isolates and seals off one ion gated channel. the only way ions can get through is if channel opens

what is the mechanism that snaps voltage gated channels closed quickly (even before membrane repolarizes)

inactivation peptide blocks flow of K+ through membrane like a tampon… this is the phase when repolarization has not happened yet but channel has closed

transporter mediated transport/facilitated transport

mediated by protein transporters that undergo a conformational change when bound by solute

uniporter

type of facilitated transporter; transport one thing at a time

symporter

type of facilitated transporter, coupled transport (2 things at once in the same direction)

antiporter

type of facilitated transporter, coupled transport (2 things at once in opposite directions)

sodium potassium pump

example of active transport pump:

• 3 Na+ ions bind —> ATP binds —> close cytoplasmic side —> open to outside —> 2K+ bind —> eventual release of K+ with ATP hydrolysis

• each ATP hydrolysis results in 3 NA+ moving outside and 2K+ moving inside

types of transport by integral membrane proteins (summary)

passive:

• direct diffusion

• channels

• facilitated diffusion by transporters

active:

• direct w/ ATP; pumps

• indirect (ie. coordinated movement of one solute up its concentration coupled to another moving down)

Na+/glucose co transporter/symporter

• indirect active transport driven by Na+ gradient across plasma membrane

• movement of glucose from low extracellular concentration to high cytosolic concentration (against gradient) driven by energy stored in Na+ gradient

sodium moves down concentration gradient while glucose moves against concentration gradient; both moving into membrane

polarized cell

apical and basolateral parts of cell have distinct protein/lipid compositions (ie. epithelial cells)

glucose transport across epithelial cell

• intestinal lumen —> epithelial cell via active transport (low to high concentration)

• epithelial cell —> blood space via diffusion (high to low concentration)

• mediated by selective localization of Na+/glucose cotransporter in apical domain and facilitated diffusion of glucose transporter/uniporter to basolateral domain

channels

passive

pumps

active

transporters

uniporters: passive

cotransporters: active

transmembrane domain of proteins must consist of ______ to have favorable interactions w/ hydrophobic tails

nonpolar amino acids

hydrophobicity plots

hydrophobic regions of ~20 amino acids indicate a transmembrane domain (can count how many there are based on graph)

what is amino acid length of hydrophobic segments

20 amino acids

how do we extract integral membrane proteins

extract using an amphipathic detergent that will dissolve bilayer

how do we extract peripheral membrane proteins

extract w/o dissolving bilayer, use high/low salt extraction

ionic detergents

solubilize and denature integral membrane proteins (ie. SDS). use if doing immunoblot after

nonionic detergent

more gentle, preserves protein structure and function while solubilizing/extracting it (ie. Triton X-100). use if want to study function (structure determines function)

*after poking holes, use differential centrifugation to remove cytosol (supernatant)

reconstitution

taking parts from a cell to study in a cell-free environment in liposomes or in vitro environment to eliminate noise and get more control

how can SDS ionic detergent work well with immunoblot

SDS is one of the early steps in preparing samples for a western/immunoblot.

• SDS denatures protein into linear form and gives it a positive charge so it can flow towards positive end of gel

• allows protein to move according to its molecular weight instead of being affected by charge

hydropathy plots

visualize amphipathic nature of integral membrane proteins

positive peaks on hydropathy plot

stretches of hydrophobic amino acids, often indicating transmembrane alpha helices

low/negative regions

hydrophilic segments exposed to cytosol or extracellular space