Lecture 36: Membranes and Membrane Proteins28/11/2011 20:55:00

Cell membranes act as selective barriers• Prevent molecules from mixing

Three roles of plasma membrane• Receiving information (signaling)• Import/export (transportation)• Motility/cell growth

Membranes enclose many different compartments in a eukaryotic cell:• Nucleus (2x)• Mitochondria (2x)• ER, vesicles, golgi apparatus, lysosome, peroxisome

The Lipid Bilayer• Two-dimensional fluid• Fluidity depends on composition• Lipid bilayer is asymmetrical• Lipid asymmetry is generated inside the cell• Hydrophilic head, hydrophobic tail• The more unsaturated the tails are, fluidity is increased• Phosphatidylcholine is the most common phospholipid in cell

membranes• There are different types of membrane lipids and all are amphipathic• Hydrophilic molecules attract water, like dissolves in like

• Hydrophobic molecules avoid water• Fats are hydrophobic, phospholipids are amphipathic (and form a

bilayer in water)• Pure phospholipids can form closed, spherical liposomes• Phospholipids can move

o Lateralo Flexiono Rotationo Flip-flop

• Fluidity depends on compositiono Cholesterol stiffenso Low temperature, less unsaturation, long tails all reduce

fluidity.• Phospho and glycolipids are distributed asymmetrically in the plasma

membraneo Glycolipids found on outsideo Phosphatidyl-serine, inositol, ethanolamine found on inside

usually.• Flippases transfer phospholipids to other side of membrane• New membranes are synthesized from ER.

o Form vesicles which fuse with other membranes

Membrane Proteins• Polypeptide chain usually crosses the bilayer as an α-helix• Proteins can be solubilized in detergents and purified

• Plasma membrane is reinforced by the cell cortex• Cell surface is coated with carbohydrate• Cells can restrict movement of membrane proteins• Functions

o Transporters (ie Na pump)o Anchors (integrins)o Receptors (platelet-derived growth factor receptor)o Enzymes (adenyl cyclase)

• 50% of mass of plasma membranes, 50 times more lipid than protein molecules.

• Different ways of associating with membrane:o Alpha helix, beta pleated sheets, transmembrane, lipid linkedo Can be peripheral (protein attached)

• Folded up proteins traverse membrane easier because the polar backbone is exposed

• Multiple alpha helixes form a hydrophilic pore• Porin proteins form water-filled channels in the outer membrane of a

bacteriumo Formed by 16 strands of β-sheetso Allows passage of ions and nutrients across outer membranes

of some bacteria and of mitochondria• Membranes are disrupted by detergents such as SDS and Triton X-100

o Have only one tail• Bacteriorhodopsin acts as a proton pump powered by light, drives ATP

synthase• Plasma membrane reinforced by cell cortex – imparts shape and

function

o Spectrin meshwork forms the cell cortex in red blood cells• Eukaryotic cells are sugar coated

o Absorb water for lubricationo Cell-cell recognitiono Protect cell from physical, chemical, enzymatic damageo Recognition of cell surface carb on neutrophils mediates

migration in infection• Movement can be restricted by cells

o Tethering to cell cortex, extracellular matrix, proteins on surface of another cell, or by barriers of diffusion like tight junctions.

Lecture 37I. General Principles of Cell Signaling

• Can act over long or short range• Each cell responds to limited set of signals• Signals relayed via intracellular signaling pathways• Nitric oxide crosses plasma membrane and activates intracellular

enzymes directly• Some hormones cross plasma membrane and bind to intracellular

receptors• There are three classes of cell surface receptors• Ion channel-linked receptors convert chemical into electrical signals• Intracellular signaling proteins act as molecular switches• Origins in unicellular organisms

o Yeast shows single cell-cell communication

o Two mating types, a and α plus a secreted mating factor signal

• Signals transduction: conversion of one type of signal into anothero Extracellular -> intracellular

• 4 ways animal cells signalo Endocrineo Paracrineo Neuronalo Contact-dependent

Lateral inhibition: Unspecified epithelial cells, one cell is dedicated to becoming a nerve cell and inhibits surrounding cells by Delta-Notch signaling.

• One signal molecule can induce different responses in different cellso ie: acetylcholine: (time scale is seconds to minutes)

In heart muscle cells, causes decreased rate and force of contraction.

In salivary gland cells, causes secretion. In skeletal muscle cells, causes contraction.

• An animal cell depends on multiple extracellular signals • Extracellular signal molecules can alter activity of diverse cell proteins

which in turn alter cellular behavioro The intracellular signaling proteins are involved in a signaling

cascade which ultimately reach the target proteins for altered behavior like metabolism, gene expression, and cell shape or movement.

• Cellular signaling cascades can follow a complex patho Primary transduction, relay, amplification, or branching to

different targets.• Extracellular signal molecules can either bind to cell surface receptors

or to intracellular enzymes or receptors (like nitric oxide)o Nitric oxide is a product of nitroglycerin which is taken to

relax smooth muscle cells.

Triggers smooth muscle relaxation in blood-vessel wall• Steroid hormones bind intracellular receptors that act as gene

regulatory proteinso Cross plasma membrane, like NOo Cholesterol does not cross membrane, rather inserts IN

membrane.o Cortisol acts by activating a gene regulatory protein

• Most signal molecules bind to receptor proteins on the target cell surface

o Extracellular domains are the cell surface receptoro Three basic classes:

Ion channel linked -> nervous system, muscle G-protein linked -> all cells Enzyme-linked -> all cells

• Many intracellular signaling proteins act as molecular switcheso Signaling by phosphorylation

Signal in by phosphorylation, off by phosphatase inactivation.

o Signaling by GTP-binding protein GTP binds to G-protein, turning it on. GTP hydrolysis inactivates by removing P.

II. G-protein-linked Receptors• Stimulation of G-protein linked receptors• G proteins can regulate ion channels

• G proteins can activate membrane bound enzymes• Cyclic AMP pathway can activate downstream genes• Inositol phospholipid pathway triggers rise in Ca• Ca signal triggers many biological processes• Intracellular signaling cascades can achieve astonishing speed (ie

photoreceptors in the eye)• All G-protein linked receptors possess a similar structure

o 7 transmembrane proteino Ligand binds to extracellular binding domaino Cytoplasmic domain which binds to G-proteino Tetramer is active, GDP can dissociate, GTP can bind, and

then complex dissociates into two activated parts.o The alpha subunit switches itself off by hydrolyzing

bound GTP• G proteins couple receptor activation to opening of cardiomyocyte K

channelso Acetylcholine binds to G protein linked receptoro Beta gamma complex binds to closed K channel to open ito Alpha subunit is inactivated (by hydrolysis) and inactive

complex reassociates with betta gamma complex to close K channel.

• Enzymes activated by G proteins catalyze synthesis of intracellular second messengers

o Alpha subunit activates adenylyl cyclase which makes lots of cylic AMP.

o Cyclic AMP concentration rises rapidly in response to neurotransmitter serotonin

o Cyclic AMP is synthesized by adenylyl cyclase, degraded by cAMP phosphodiesterase

• Extracellular signals can act rapidly or slowly• Rise in intracellular cyclic AMP can activate gene transcription through

protein kinase A

o Translocates through nuclear pore, into nucleus, phosphorylates gene regulatory protein to activate target gene.

• Membrane bound phospholipase C activates two small messenger molecules: IP3, DAG

o Phospholipase C activated by alpha subunit, splits inositol phospholipid into IP3 and DAG

o IP3 opens Ca channel in ER, Ca is released and works with DAG to activate Protein Kinase C.

• Fertilization of an egg by sperm triggers a rapid increase in cytosolic Cao Other processes triggered by Ca signal:

Sperm entry -> embryonic development Skeletal muscle -> contraction Nerve cells -> secretion

• Calcium/Calmodulin complex are what bind to proteins.• A rod photoreceptor cell from the retina is exquisitely sensitive to light

o G protein linked light receptor activates G protein transducing, activated alpha subunit causes Na channels to close.

o Light induced signaling cascade in rod photoreceptors greatly amplifies light signals.

III. Enzyme linked receptors• Activated receptor tyrosine kinases assemble a complex of intracellular

signaling proteinso Ligand brings two tyrosine kinase domains together,

phosphorylated to activate. Intracellular signaling proteins bind to phosphorylated tyrosines.

o Activated complex includes Ras-activating protein, which is anchored in membrane, transmits signal downstream.

Ras is monomeric GTP-binding protein, not a trimeric G protein, but resembles the alpha subunit and functions as a molecular switch.

30% of cancers arise from mutations in Ras. Ras activates a MAP-kinase phosphorylation cascade

• Some enzyme-linked receptors activate a fast track to the nucleus• Protein kinase networks integrate info to control complex cell behaviors• Multicellularity and cell communication evolved independently in plants

and animals• Cytokine receptors are associated with cytoplasmic tyrosine kinases

o JAK kinases phosphorylate receptor which recruits cytoplasmic proteins.

• TGF-beta/BMP receptors activate gene regulatory proteins directly at the plasma membrane

• Signaling pathways can be highly interconnected: cross-talk

Lecture 38General introduction:

• Membrane enclosed organelles are distributed throughout the cytoplasm

o Thousands of different reactions occur simultaneously, are partitioned

o Cytosol is 54% of cello Mitochondria is 22% of cellso ER is 12% of cell (1 per cell)

• Nuclear membrane and ER may have evolved at the same time through invagination of plasma membrane.

• Mitochondria are thought to have originated from aerobic prokaryote being engulfed by a larger anaerobic eukaryotic cell -> has it’s own genome.

• Nucleus is a double membrane organelleo Encloses nuclear DNA, defines nuclear compartment and

contains most of the genetic information.

o Export, import through nuclear pore complex Contains about 100 proteins, two way gate, export of

mRNA, and ribosome subunits. Import of proteins requires a signal sequence called the

nuclear localization signal Requires energy (GTP) and special chaperone proteins Export of RNA from nucleus – RNA molecules are made

in the nucleus and exported to the cytoplasm as processed mRNA

• One Endoplasmic Reticulumo System of interconnected sacs and tubes of membraneo Extend throughout most of cello Major site of new membrane (lipid) synthesiso With ribosomes on cytosolic side = rough ERo Without ribosomes = smooth ERo Most extensive network membrane in eukaryotic cells

• Golgi apparatuso Flattened sacs called cisternae which are piled like stacks of

plateso Usually near nucleuso Two faces:

Cis face adjacent to ER Trans face towards plasma membrane (where post

translational modification occurs)o Receives proteins and lipidso Site of modification of proteins and lipidso Dispatches proteins and lipids to final destinations

o Transport vesicles bud off• Other membrane enclosed organelles

o Endosomes – small membrane enclosed organelles that sort ingested molecules in endocytosed materials. Passed to lysosomes or recycled back to the plasma membrane.

o Lysosomes – small sacs containing digestive enzymes that degrade organelles, macromolecules, and particles taken in by endocytosis. “garbage disposal of the cell.” Ph about 7.2

o Peroxisomes – small membrane enclosed organelle containing oxidative enzymes that break down lipids and destroy toxic molecules

• Protein transporto Multiple modes of protein transport (import and export)o Three mechanisms

Transport through nuclear pores: protein with nuclear localization signal enter through pores

Across membranes: proteins moving from cytosol into ER, mitochondria and peroxisomes transported across organelle membrane by protein translocators

By vesicles: from ER onward and from one endomembrane compartment to another ferried by transport vesicles

• Protein sorting signalso Specific amino acid sequenceo Directs protein to organelleo Proteins without signals remain in cytosolo Signal sequences direct proteins to different compoartments

Continuous stretch of AA usually 15-20 residues in length

Usually removed after the protein reaches destination Organelles and signal sequences:

ER import rich in V A L I and retendtion KDEL Mitochondria rich in R

Nucleus PPKKKRKV Peroxisomes SKL

o Signal sequences are both necessary and sufficient to direct protein to organelles

• ER: entry point for protein distributiono Proteins destined for golgi, lysosomes, endosomes and cell

surfaces first enter ER from cytosolo Once inside ER or membrane, proteins do not reenter cytosolo Water soluble proteins are completely translocated across ER

membrane and released into ER lumeno Transmembrane proteins only partially translocated across ER

membrane and become embedded• Vesicular transport

o Entry into ERo To golgi apparatuso From er -> golgi -> other by continuous budding, fusion of

transport vesicleso Vesicle transport provides routes of communication

• Protein transport: quality controlo Most proteins that enter ER are destined for other locationso Exit from the ER is highly selective: improperly modified and

or folded proteins are retained in lumen; dimeric or multimeric proteins that fail to assemble are also retained

• Exocytosiso Constitutive: newly synthesized proteins, lipids, and carbs

delivered from ER via golgi to subcellular locations, extracellularly to ECM via transport vesicles.

Lipids and proteins supplied to plasma membrane Proteins secreted into ECM or onto the cell surface

o Regulated

Specialized secretory cells synthesize high levels of proteins such as hormones or digestive enzymes that are stored in secretory vesicles for subsequent release

Vesicles bud off from trans golgi network and accumulate adjacent to plasma membrane until mobilized by extracellular signal

• Endocytosiso Pinocytosis (drinking)

Internalizes plasma membrane: as much membrane is added to cell surface by exocytosis as is removed by endocytosis – total surface area and volume remain unchanged.

Mainly carried out by transport vesicles: deliver extracellular fluid and solutes to endosomes; fluid intake is balanced by fluid loss during exocytosis

o Phagocytosis (eating) Specialized cells only

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Lecture 39: Cytoskeleton

Roles of cytoskeletal filaments:• Intermediate – cell structure against mechanical stress• Microtubules – intracellular transport, railroad of cell• Actin – membrane mobility; cell movement

Intermediate filaments:• 10 nm in diameter• Rope like structure composed of long polypeptides twisted together• Associated with cell junctions• Mechanical strength, cell shape, cell-cell contacts, and structure for

nuclear envelope• Monomers -> dimer -> tetramer -> 8 tetramers make one ropelike

filament• Different proteins:

o Epithelia – keratinso Connective tissue, muscles, neuroglial cells – vimentino Nerve cells – neurofilaments o Nuclear envelope in animal cells – nuclear lamins

• Mutation in keratin genes = epidermolysis bullosa simplex• Networks of filaments connect across desmosomes in epithelia

Microtubules:• 25 nm wide• Hollow, made of α and β tubulin anchored to γ tubulin• Have polarity – gives directionality• “Dynamic instability”: built or disassembled as needed

o Zip up to growo Unravel and tubulin molecules fall off if not neededo This is done by GTP since tubulin are GTPases

GTPases are the cell’s timers High energy phosphate bond. Molecules with GTPases

hydrolyze that bond, leaving GDP GTP between α and β tubulin molecules makes them

straighter, so they pack better. GTP hydrolysis makes them kinked, so they fall off.

• Organize cell organelles and control traffic of vesicles• Roles in interphase cell, dividing cell, ciliated cells, flagella.• The centrosome

o Centriles inside of centrosome, nobody knows what they doo Centrosome is an envelope of tubulin where microtubules

extend out with plus end out.• Structure:

o α and β tubulin strands • Stabilizing or destabilizing MTs

o Microtubule associated proteins (MAPs) Bind to free ends of MTs and stabilize ends selectively

to polarize a cello Drugs can be used to change MT stability

Colchicine binds free tubulin to prevent polymerization; MTs disintegrate and mitosis stops

Taxol prevents loss of subunits from MTs; MTs become “frozen” in place and mitosis stops.

• MT organized transporto Anterograde transport, retrograde transport.

• Motor proteins use ATP to power transport along the MT railroado Kinesin and dynein are dimers that walk along microtubule.o One ATP is used per step.

• Cilia and flagella are made of MTs

Actin Filaments:• Control cell movement• Found in:

o Epithelial cell microvillio Stress fibers in cultured cellso Leading edge lamellipodiao Contractile ring in dividing cells – cytokinesis

• Actin polymerization requires ATPo Free G-actin monomers use ATP to become F-actin to form

filaments. To uncoil, hydrolyze ATP and fall apart.• Actin dynamics provide force for membrane movement

o ARP complex create branches

o Depolymerizing protein promotes ATP hydrolysiso Capping proteins cap the ends and stabilize ATP bound

monomer, stabilizing leading edge.• Actin binding proteins link actin fibers to the membrane and other

cellular components• Integrins link actin to focal adhesions

o Binds to extracellular structures, messages to actin.• Cells move by actin crawling (dynamics)• Axon growth cone crawling• Rho family GTPases control actin dynamics

o RhoA causes stress fibers Stabilize actin filaments Induces myosin phosphorylation and thus contractility

o Cdc432 causes filopodia extension Promotes actin nucleating by ARP complexes

o Rac promotes lamellipodia extension Promotes actin nucleation, but also uncapping to allow

more sites of nucleationo Cell surface receptors modulate Rho family activity

Attractive cues activate Rac and Cdc42 on area of growth cone

Repulsive cues activate RhoA Growth cone turns

• Myosins: actin motor proteinso Head, neck, tailo Tails link up togethero Work as dimerso Moves membranes or cell components

• Muscle contraction by actin and myosino Myosin heads climb up actin filamento Z disks move together, muscle contracts